Hemagglutinin and fusion polypeptide of canine distemper virus

Abstract

The present specification provides methods and materials related to CDV H and / or CDV F polypeptides, such as CDV H polypeptides, CDV F polypeptides, recombinant viruses (e.g., vesicular stomatitis virus (VSV)) containing CDV H and / or CDV F polypeptides, nucleic acid molecules encoding CDV H and / or CDV F polypeptides, methods of producing recombinant viruses (e.g., VSV) containing CDV H and / or CDV F polypeptides, and methods of using recombinant viruses (e.g., VSV) containing CDV H and / or CDV F polypeptides to treat cancer or infectious diseases.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 62 / 913,111, filed on 9 October 2019. The disclosures of the prior application are deemed to be part of the disclosures of this application (and are incorporated into the disclosures of this application by reference). Sequence List This application includes a sequence listing, which is submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on November 25, 2020, is named 07039-1936WO1_SL.txt and has a size of 1,096,580 bytes.

[0002] 1. Field This specification relates to canine distemper virus (CDV) hemagglutinin (H) and fusion (F) polypeptides. For example, this specification relates to CDV H polypeptide, CDV F polypeptide, recombinant viruses containing CDV H polypeptide and / or CDV F polypeptide (e.g., vesicular stomatitis virus (VSV)), nucleic acid molecules encoding CDV H polypeptide and / or CDV F polypeptide, methods for producing recombinant viruses containing CDV H polypeptide and / or CDV F polypeptide (e.g., VSV), and methods for using recombinant viruses containing CDV H polypeptide and / or CDV F polypeptide (e.g., VSV) to treat cancer or infection. [Background technology]

[0003] 2. Background information Viruses such as VSV, measles virus (MeV), and adenovirus can be used as oncolytic viruses to treat cancer. Vesicular stomatitis virus (VSV) is a virus belonging to the Rhabdoviridae family. The VSV genome is a single-molecule negative-strand RNA that encodes five major polypeptides: nucleocapsid (N) polypeptide, phosphorylated protein (P) polypeptide, matrix (M) polypeptide, glycoprotein (G) polypeptide, and viral polymerase (L) polypeptide. [Overview of the project]

[0004] overview This specification provides methods and materials relating to CDV H and / or CDV F polypeptides. For example, this specification provides CDV H polypeptide, CDV F polypeptide, recombinant viruses containing CDV H polypeptide and / or CDV F polypeptide (e.g., vesicular stomatitis virus (VSV)), nucleic acid molecules encoding CDV H polypeptide and / or CDV F polypeptide, methods for producing recombinant viruses containing CDV H polypeptide and / or CDV F polypeptide (e.g., VSV), and methods for using recombinant viruses containing CDV H polypeptide and / or CDV F polypeptide (e.g., VSV) to treat cancer or infection.

[0005] As described herein, CDV F polypeptides can be designed to exhibit increased fusion activity when expressed in cells in combination with CDV H polypeptides, compared to the level of fusion activity of wild-type CDV F polypeptides expressed in equivalent cells in combination with the CDV H polypeptide. For example, a CDV F polypeptide designed to have a truncated signal peptide sequence may exhibit increased fusion activity when expressed in cells in combination with a CDV H polypeptide (e.g., wild-type or detargeted CDV H polypeptide), compared to the level of fusion activity of wild-type CDV F polypeptides containing the full-length signal peptide sequence expressed in equivalent cells in combination with the CDV H polypeptide. Such CDV F polypeptides can be incorporated into viruses to create recombinant viruses that have the ability to increase the fusion activity observed in cells infected with the virus.

[0006] Furthermore, as described herein, CDV H polypeptides can be designed to be detargeted so as not to have the ability to enter or fuse with cells via Nectin-4 polypeptide, SLAMF1 polypeptide, or viral receptors present on wild-type Vero cells when used in combination with F polypeptides (e.g., CDV F polypeptides). Such CDV H polypeptides can provide a platform for designing H polypeptides that have the ability to retarget one or more targets of interest. For example, the H polypeptides described herein can be further modified to include a binding sequence (e.g., a single-chain antibody (scFv) sequence) that has binding specificity to a target of interest, so that recombinant viruses containing the retargeted H polypeptides and F polypeptides can infect cells expressing their targets.

[0007] Furthermore, viruses such as VSV can be designed to have nucleic acid molecules encoding VSV N polypeptide, VSV P polypeptide, VSV M polypeptide, CDV F polypeptide (e.g., wild-type CDV F polypeptide or a modified CDV F polypeptide as described herein), CDV H polypeptide (e.g., wild-type CDV H polypeptide or a modified CDV H polypeptide as described herein), and VSV L polypeptide. Such nucleic acid molecules may lack a functional VSV G polypeptide and / or a nucleic acid sequence encoding a full-length VSV G polypeptide. For example, the VSV described herein can be designed to have a nucleic acid sequence encoding VSV N polypeptide, VSV P polypeptide, VSV M polypeptide, CDV F polypeptide (e.g., wild-type CDV F polypeptide or a modified CDV F polypeptide as described herein), CDV H polypeptide (e.g., wild-type CDV H polypeptide or a modified CDV H polypeptide as described herein), and VSV L polypeptide, but lacking the ability to encode a functional VSV G polypeptide. In some cases, the VSVs described herein can be designed to have nucleic acid molecules encoding VSV N polypeptide, VSV P polypeptide, VSV M polypeptide, CDV F polypeptide (e.g., wild-type CDV F polypeptide or a modified CDV F polypeptide described herein), CDV H polypeptide (e.g., wild-type CDV H polypeptide or a modified CDV H polypeptide described herein), and VSV L polypeptide, wherein the nucleic acid sequences encoding the CDV F polypeptide and CDV H polypeptide are located at the positions where the nucleic acid sequence encoding the full-length VSV G polypeptide is normally located in wild-type VSV.In some cases, the VSV described herein can be designed to have a nucleic acid molecule in which the nucleic acid sequence encoding the VSV G polypeptide is replaced with a nucleic acid encoding the CDV F polypeptide (e.g., wild-type CDV F polypeptide or a modified CDV F polypeptide described herein) and the CDV H polypeptide (e.g., wild-type CDV H polypeptide or a modified CDV H polypeptide described herein).

[0008] As described herein, the VSV / CDV hybrid can be designed to have CDV selectivity and rapid replication as seen in wild-type or parental VSV. In some cases, the VSV / CDV hybrid described herein can be designed to have a preselected tropism. For example, CDV F and / or H polypeptides with knocked-out specificity for Nectin-4 and / or SLAMF1 can be used. In such cases, an scFv or polypeptide ligand can be attached, for example, to the C-terminus of the CDV H polypeptide. In that case, the scFv or polypeptide ligand can determine the tropism of the VSV / CDV hybrid. Examples of scFvs that can be used to direct the VSV / CDV hybrid towards a cellular receptor (e.g., a tumor-associated cellular receptor) include, but are not limited to, anti-EGFR, anti-αFR, anti-CD46, anti-CD38, anti-HER2 / neu, anti-EpCAM, anti-CEA, anti-CD20, anti-CD133, anti-CD117 (c-Kit), and anti-CD138 and anti-PSMA scFvs. Examples of polypeptide ligands that can be used to direct the VSV / CDV hybrid include, but are not limited to, the urokinase plasminogen activator uPA polypeptide, cytokines such as IL-13 or IL-6, single-chain T cell receptors (scTCRs), exatin polypeptides, stem cell factor (SCF), EGF, and integrin-binding polypeptides.

[0009] In some examples, the VSV / CDV hybrids described herein may have nucleic acid molecules containing sequences encoding interferon (IFN) polypeptides (e.g., human IFN-β polypeptide), sodium iodide cotransporter (NIS) polypeptides (e.g., human NIS polypeptide), fluorescent polypeptides (e.g., GFP polypeptide), any suitable therapeutic transgene (e.g., HSV-TK or cytosine deaminase), polypeptides that antagonize host immunity (e.g., influenza NS1, HSVγ34.5, or SOCS1), or tumor antigens (e.g., cancer vaccine components). The nucleic acid encoding the IFN polypeptide can be positioned between the nucleic acid encoding the VSV M polypeptide and the nucleic acid encoding the VSV L polypeptide. Such a position allows the virus to express an amount of IFN polypeptide sufficient to activate an antiviral innate immune response in non-cancer tissues without interfering with efficient viral replication in cancer cells, and thus can mitigate potential virogenicity. The nucleic acid encoding the NIS polypeptide can be positioned between the nucleic acid encoding the VSV M polypeptide and the VSV L polypeptide. Such positions allow the virus to express an amount of NIS polypeptide sufficient to enable selective accumulation of iodine in infected cells, thereby enabling both imaging of viral distribution using radioisotopes and radiotherapy targeting infected cancer cells, and (b) allow the virus to express an amount of NIS polypeptide that is not high enough to be toxic to infected cells. Placing the nucleic acid encoding the IFN polypeptide between the nucleic acid encoding the VSV M polypeptide and the nucleic acid encoding the VSV L polypeptide, and placing the nucleic acid encoding the NIS polypeptide between the nucleic acid encoding the VSV M polypeptide and the VSV L polypeptide within the VSV genome can result in a VSV that is viable, capable of replicating and spreading, expresses an appropriate level of functional IFN polypeptide, and further expresses an appropriate level of functional NIS polypeptide for taking up radioiodine for imaging and radiotherapy.

[0010] Generally, certain aspects of the present specification feature a CDV F polypeptide having a signal peptide sequence less than 75 amino acid residues in length. The signal peptide sequence can include 75 or fewer amino acid residues of SEQ ID NO: 6. The CDV F polypeptide can include SEQ ID NO: 4, provided that the CDV F polypeptide lacks at least amino acid residues 1-60 of SEQ ID NO: 4, or lacks at least amino acid residues 1-105 of SEQ ID NO: 4. A recombinant virus comprising this CDV F polypeptide and a CDV H polypeptide can exhibit increased fusion activity compared to an equivalent control recombinant virus comprising a full-length wild-type CDV F polypeptide and a CDV H polypeptide.

[0011] In another embodiment, the present specification features a nucleic acid molecule encoding a CDV F polypeptide. The CDV F polypeptide can have a signal peptide sequence less than 75 amino acid residues in length. The signal peptide sequence can include 75 or fewer amino acid residues of SEQ ID NO: 6. The CDV F polypeptide can include SEQ ID NO: 4, provided that the CDV F polypeptide lacks at least amino acid residues 1-60 of SEQ ID NO: 4, or lacks at least amino acid residues 1-105 of SEQ ID NO: 4. A recombinant virus comprising this CDV F polypeptide and a CDV H polypeptide can exhibit increased fusion activity compared to an equivalent control recombinant virus comprising a full-length wild-type CDV F polypeptide and a CDV H polypeptide.

[0012] In another embodiment, this specification features a recombinant virus comprising the CDV F polypeptide. The CDV F polypeptide may have a signal peptide sequence less than 75 amino acid residues in length. The signal peptide sequence may contain 75 or fewer amino acid residues of SEQ ID NO: 6. The CDV F polypeptide may contain SEQ ID NO: 4, provided that the CDV F polypeptide lacks at least amino acid residues 1-60 of SEQ ID NO: 4, or at least amino acid residues 1-105 of SEQ ID NO: 4. Recombinant viruses comprising this CDV F polypeptide and CDV H polypeptide may exhibit increased fusion activity compared to equivalent control recombinant viruses comprising full-length wild-type CDV F polypeptide and CDV H polypeptide.

[0013] In another embodiment, this specification features a recombinant virus comprising a nucleic acid molecule. The nucleic acid molecule may encode a CDV F polypeptide. The CDV F polypeptide may have a signal peptide sequence less than 75 amino acid residues in length. The signal peptide sequence may contain 75 or fewer amino acid residues of SEQ ID NO: 6. The CDV F polypeptide may contain SEQ ID NO: 4, provided that the CDV F polypeptide lacks at least amino acid residues 1-60 of SEQ ID NO: 4, or at least amino acid residues 1-105 of SEQ ID NO: 4. Recombinant viruses comprising this CDV F polypeptide and CDV H polypeptide may exhibit increased fusion activity compared to equivalent control recombinant viruses comprising full-length wild-type CDV F polypeptide and CDV H polypeptide.

[0014] In another embodiment, this specification features CDV H polypeptides containing 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, 548A, or combinations thereof, according to the amino acid numbering of Sequence ID No. 2. The CDV H polypeptide may contain combinations of two, three, four, five, or six of 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, and 548A. CDV H polypeptides can contain combinations of 7, 8, 9, 10, or 11 of 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, and 548A. CDV H polypeptides can contain combinations of 12, 13, or 14 of 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, and 548A. CDV H polypeptides can contain 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, and 548A. CDV H polypeptides can also contain M437 according to the amino acid numbering of SEQ ID NO: 5.

[0015] In another embodiment, this specification features a CDV H polypeptide comprising the sequence shown in Figure 11, except that the sequence includes mutations in presented amino acid residues selected from the group consisting of P454, V / L / F460, L / F / W479, I494, I / L / V510, Y520, Y / N525, D / G526, I / V527, S / T528, R529, Y / D537, Y539, Y / F547, and T / M548 according to the amino acid numbering of SEQ ID NO: 5. The CDV H polypeptide may include mutations in 2, 3, 4, 5, or 6 presented amino acid residues selected from the group. The CDV H polypeptide may include mutations in 7, 8, 9, 10, or 11 presented amino acid residues selected from the group. The CDV H polypeptide may include mutations in 12, 13, or 14 presented amino acid residues selected from the group. CDV H polypeptides can contain mutations in their group of presented amino acid residues. CDV H polypeptides can contain M437, according to the amino acid numbering in SEQ ID NO: 5.

[0016] In another embodiment, this specification features a nucleic acid molecule encoding a CDV H polypeptide. The CDV H polypeptide may include the sequence shown in Figure 11, except that the sequence includes mutations in presented amino acid residues selected from the group consisting of P454, V / L / F460, L / F / W479, I494, I / L / V510, Y520, Y / N525, D / G526, I / V527, S / T528, R529, Y / D537, Y539, Y / F547, and T / M548 according to the amino acid numbering of SEQ ID NO: 5. The CDV H polypeptide may include mutations in 2, 3, 4, 5, or 6 presented amino acid residues selected from that group. The CDV H polypeptide may include mutations in 7, 8, 9, 10, or 11 presented amino acid residues selected from that group. The CDV H polypeptide may include mutations in 12, 13, or 14 presented amino acid residues selected from that group. CDV H polypeptides can contain mutations in their group of presented amino acid residues. CDV H polypeptides can contain M437, according to the amino acid numbering in SEQ ID NO: 5.

[0017] In another embodiment, this specification features a recombinant virus comprising the CDV H polypeptide. The CDV H polypeptide is a polypeptide comprising the sequence shown in Figure 11, except that the sequence comprises mutations of presented amino acid residues selected from the group consisting of P454, V / L / F460, L / F / W479, I494, I / L / V510, Y520, Y / N525, D / G526, I / V527, S / T528, R529, Y / D537, Y539, Y / F547, and T / M548 according to the amino acid numbering of SEQ ID NO: 5. The CDV H polypeptide may contain mutations of 2, 3, 4, 5, or 6 presented amino acid residues selected from the group. The CDV H polypeptide may contain mutations of 7, 8, 9, 10, or 11 presented amino acid residues selected from the group. The CDV H polypeptide may contain mutations in 12, 13, or 14 presenting amino acid residues selected from a group. The CDV H polypeptide may contain mutations in a group of presenting amino acid residues. The CDV H polypeptide may contain M437 according to the amino acid numbering of SEQ ID NO: 5. The virus may contain the CDV F polypeptide, which contains a signal peptide sequence less than 75 amino acid residues in length. The signal peptide sequence may contain 75 or fewer amino acid residues of SEQ ID NO: 6. The CDV F polypeptide may contain SEQ ID NO: 4, provided that the CDV F polypeptide lacks at least amino acid residues 1-60 of SEQ ID NO: 4, or at least amino acid residues 1-105 of SEQ ID NO: 4. Recombinant viruses containing this CDV F polypeptide and CDV H polypeptide may exhibit increased fusion activity compared to equivalent control recombinant viruses containing full-length wild-type CDV F polypeptide and CDV H polypeptide.

[0018] In another embodiment, this specification features a recombinant virus comprising a nucleic acid molecule encoding the CDV H polypeptide. The CDV H polypeptide may include the sequence shown in Figure 11, except that the sequence includes mutations in presented amino acid residues selected from the group consisting of P454, V / L / F460, L / F / W479, I494, I / L / V510, Y520, Y / N525, D / G526, I / V527, S / T528, R529, Y / D537, Y539, Y / F547, and T / M548 according to the amino acid numbering of SEQ ID NO: 5. The CDV H polypeptide may include mutations in 2, 3, 4, 5, or 6 presented amino acid residues selected from that group. The CDV H polypeptide may include mutations in 7, 8, 9, 10, or 11 presented amino acid residues selected from that group. The CDV H polypeptide may contain mutations in 12, 13, or 14 present amino acid residues selected from a set. The CDV H polypeptide may contain mutations in a set of present amino acid residues. The CDV H polypeptide may contain M437 according to the amino acid numbering of SEQ ID NO: 5. The virus may contain a nucleic acid molecule encoding the CDV F polypeptide. The CDV F polypeptide may have a signal peptide sequence less than 75 amino acid residues in length. The signal peptide sequence may contain 75 or fewer amino acid residues of SEQ ID NO: 6. The CDV F polypeptide may contain SEQ ID NO: 4, provided that the CDV F polypeptide lacks at least amino acid residues 1-60 of SEQ ID NO: 4, or at least amino acid residues 1-105 of SEQ ID NO: 4. Recombinant viruses containing these CDV F polypeptides and CDV H polypeptides may exhibit increased fusion activity compared to equivalent control recombinant viruses containing full-length wild-type CDV F polypeptides and CDV H polypeptides.

[0019] In another embodiment, this specification features (a) CDV and (b) a recombinant virus described herein, which is a hybrid virus of VSV, MeV, or adenovirus.

[0020] In another embodiment, this specification features a replicable vesicular stomatitis virus comprising an RNA molecule, the RNA molecule comprising a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV N polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV P polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV M polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV F polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV H polypeptide, and a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV L polypeptide, but the RNA molecule lacks a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a functional VSV G polypeptide. The CDV F polypeptide may have a signal peptide sequence less than 75 amino acid residues in length. The signal peptide sequence may contain 75 or fewer amino acid residues of SEQ ID NO: 6. The CDV F polypeptide may contain SEQ ID NO: 4, provided that the CDV F polypeptide lacks at least amino acid residues 1-60 of SEQ ID NO: 4, or at least amino acid residues 1-105 of SEQ ID NO: 4. Recombinant viruses containing the CDV F polypeptide and CDV H polypeptide can exhibit increased fusion activity compared to equivalent control recombinant viruses containing full-length wild-type CDV F polypeptide and CDV H polypeptide. The CDV H polypeptide may be the CDV H polypeptide described in one of the paragraphs above. The CDV H polypeptide may contain the amino acid sequence of a single-chain antibody. The single-chain antibody may be a single-chain antibody against CD19, CD20, CD38, CD46, EGFR, αFR, HER2 / neu, or PSMA. The RNA molecule may contain a nucleic acid sequence that serves as a template for a positive-strand transcript encoding the NIS polypeptide.

[0021] In another embodiment, this specification features a composition comprising any of the viruses described in the above paragraphs.

[0022] In another embodiment, this specification features a nucleic acid molecule comprising a nucleic acid strand containing a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV N polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV P polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV M polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV F polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV H polypeptide, and a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV L polypeptide, wherein the nucleic acid strand lacks a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a functional VSV G polypeptide. The CDV F polypeptide may have a signal peptide sequence less than 75 amino acid residues in length. The signal peptide sequence may contain 75 or fewer amino acid residues of SEQ ID NO: 6. The CDV F polypeptide may contain SEQ ID NO: 4, provided that the CDV F polypeptide lacks at least amino acid residues 1-60 of SEQ ID NO: 4, or at least amino acid residues 1-105 of SEQ ID NO: 4. Recombinant viruses containing the CDV F polypeptide and CDV H polypeptide can exhibit increased fusion activity compared to equivalent control recombinant viruses containing full-length wild-type CDV F polypeptide and CDV H polypeptide. The CDV H polypeptide may be the CDV H polypeptide described in one of the paragraphs above. The CDV H polypeptide may contain the amino acid sequence of a single-chain antibody. The single-chain antibody may be a single-chain antibody against CD19, CD20, CD38, CD46, EGFR, αFR, HER2 / neu, or PSMA. The RNA molecule may contain a nucleic acid sequence that serves as a template for a positive-strand transcript encoding the NIS polypeptide.

[0023] In another embodiment, this specification features a composition comprising any of the nucleic acid molecules described in the above paragraphs.

[0024] In another embodiment, this specification features a method for treating cancer. The method comprises administering a composition described herein (for example, a composition containing a virus described herein) to a mammal having cancer cells, wherein the number of cancer cells in the mammal decreases after administration. The mammal may be a human. The cancer may be myeloma, melanoma, glioma, lymphoma, mesothelioma, lung cancer, brain tumor, gastric cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, ovarian cancer, breast cancer, pancreatic cancer, liver cancer, or head and neck cancer.

[0025] In another embodiment, this specification features a method for inducing tumor regression in a mammal. The method comprises administering a composition described herein (for example, a composition containing a virus described herein) to a mammal having a tumor, wherein the size of the tumor is reduced after administration. The mammal may be a human. The cancer may be myeloma, melanoma, glioma, lymphoma, mesothelioma, lung cancer, brain tumor, gastric cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, ovarian cancer, breast cancer, pancreatic cancer, liver cancer, or head and neck cancer.

[0026] In another embodiment, this specification features a method for rescuing a replicable vesicular stomatitis virus from cells. The vesicular stomatitis virus comprises an RNA molecule containing a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV N polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV P polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV M polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV F polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV H polypeptide, and a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV L polypeptide, the RNA molecule lacking a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a functional VSV G polypeptide. The method comprises (a) inserting a nucleic acid encoding an RNA molecule into cells under conditions that produce a replicable vesicular stomatitis virus, and (b) recovering the replicable vesicular stomatitis virus.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the invention pertains. The invention can be carried out using methods and materials similar to or equivalent to those described herein, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict, including definitions, this specification shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to limit the invention.

[0028] Details of one or more embodiments of the present invention are described in the accompanying drawings and the following description. Other features, purposes, and advantages of the present invention will be apparent from the description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0029] [Figure 1] Molecular phylogenetic analysis of the hemagglutinin gene for CDV genotypes using the maximum likelihood method. Phylogenetic trees were estimated using the maximum likelihood method based on a generalized time-reversal model. The analysis targeted 119 CDV H total nucleotide sequences. 22458 / 15 strains and 5804 strains were classified into the Artic-like genotype and the Europe-1 / South America 1 genotype, respectively. Evolutionary analysis was performed using MEGA7. [Figure 2-1]Construction of CDV H / F complexes for target cell fusion. (A) Syncytium formation assay: Monolayers of Vero cells and Vero cells expressing human Nectin-4 polypeptide or canine SLAMF1 polypeptide were simultaneously transfected as directed with expression plasmids encoding H and F polypeptides from CDV5804, CDV22458 / 16, or CDVOnderstepoort (large plaque-forming) (CDVOL). Syncytium formation was recorded 24 hours after Giemsa staining. (B) Cells were further transfected with a GFP expression plasmid for increased sensitivity. DAPI was used for nuclear staining. (C) Schematic diagram of receptor-expanded morbillivirus adhesion protein. The receptor-binding protein contained a cytoplasmic tail (C), a transmembrane domain (T), and an ectodomain fused to a His tag following a CD38-specific single-stranded variable fragment (scFv). (D) Surface expression of receptor-expanded morbillivirus adhesion protein. HEK293T cells were transfected with a specified adhesion protein retargeted to the CD38 receptor, and surface expression was analyzed by FACS using a PE-conjugated anti-HIS antibody. (E) Schematic diagram of a quantitative fusion assay based on a self-associating split luciferase assay. Effector cells were transfected with plasmids encoding H and F polypeptides, as well as plasmids encoding one half each of sea urchin luciferase (RL) and green fluorescent protein (GFP). Target cells with receptors were transfected with a plasmid encoding the other half of the dual-split reporter gene and mixed with the effector cells. Mixing of the contents restored the enzymatic activity of the otherwise non-functional dual-split reporter gene, and this activity was measured in real time. (F) Cell-targeted fusion activity of a morbillivirus adhesion protein extended to the CD38 receptor. Effector cells were simultaneously transfected with the indicated adhesion / fusion polypeptide pair. After co-culture with target cells, the luminescence signal was recorded. The values ​​and error bars (SD) are derived from one representative experiment conducted in at least three consecutive cycles.Of particular note is the substitution of the CDV F signal peptide with the MEV homologous peptide (MeV SP). Statistical significance was calculated by two-way ANOVA using Turkey's multiple comparison test.*, p<.02;***, p<0.0005;****, p<.0001. (G) Integrity of CDV H polypeptide. HEK293T cells were transfected with the indicated CDV H polypeptide, and cell lysates were immunoblotted with anti-HIS antibody (CDV H) or anti-β-actin (loading control). (H) Cell-targeted fusion of retargeted H / F complexes. Cell fusion was measured as described in (C). Effector cells were simultaneously transfected with receptor-blind CD38 retargeted pair MeV H / F and CDV H5804 / F22458 / 16 and overlaid with CHO cells expressing the relevant receptors. Values ​​and error bars (SD) are obtained from one representative experiment performed in at least three consecutive cycles. (I) Retargeting of the CDV H / F complex to Her2 / neu. Cell fusion assays were performed using CDV H / F complexes displaying Her2 / neu-specific scFv or aphibodies (ZX). This shows the binding affinity to the displayed ligand. (J) Relationship between receptor expression and receptor binding affinity. Fusion activity of CDV H polypeptides displaying Her2 / neu-specific aphibodies with different affinities was evaluated in Her2 / neu-positive cell lines: HT1080 (3.4 × 10³ molecules / cell), Sko3pi (4.19 × 10³ molecules / cell), TET67L (1.5 × 10⁵ molecules / cell). Cells transfected with CDV F alone were used as a negative control. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 2-3] This is a continuation of Figure 2-2. [Figure 2-4] This is a continuation of Figure 2-3. [Figure 2-5] This is a continuation of Figure 2-4. [Figure 3-1]The CDV H / F complex can replace the measles virus envelope glycoprotein, leading to targeted cell entry and resistance to anti-measles virus antibodies. (A) Expression on the cell surface. CHO cells were transfected with one of the DSP plasmids and an F polypeptide (MeV or CDV F224568 / 16) expression plasmid, along with the indicated H polypeptide expression plasmid (HIS-tagged). After 24 hours, H expression was measured by CELISA using an anti-HIS antibody. (B) CHO cells transfected as in (A) were co-cultured with the indicated CHO cell-derived cell line, and luminescence values ​​were obtained over 9 hours. Values ​​and error bars (SD) were obtained from one representative experiment performed in at least three consecutive cycles. (C) Protein composition of measles virus encoding the CDV H / F complex targeted against CD46 (scFv A09, Stealth 2.0). 1.6E4 TCID50 particles were subjected to SDS-PAGE and immunoblotted with the relevant antibody. Measles virus was used as a control. (D) Growth kinetics. The growth kinetics of Stealth 2.0 virus were measured on Vero and Vero-HIS (multiple infection units (MOI) = 0.03) at specified time points. The growth kinetics of MeV on Vero / hSLAMF1 cells were included for comparison. (E) Neutralization assay. Fluorescence focus reduction neutralization assays were performed using antisera of MeV and CDV. Antisera of different dilutions were pre-incubated with a fixed amount of virus at 37°C for 1 hour. Then, Vero-HIS cells were infected with this mixture, and the viral load in the control well without antibody was set to 100%. All neutralization curves represent the mean and SD of curves obtained by performing four replicates on the same 96-well plate. (F) Receptor selectivity of recombinant virus. CHO cells expressing the relevant receptors were infected with recombinant virus at the indicated MOI. GFP autofluorescence was recorded after 2 days. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 4-1]Oncolytic activity of CD46-targeting envelope chimeric MeV. Human myeloma cell line U266.B1 was subcutaneously transplanted into CDB17 SCID mice. When tumor volume reached 500 mm3, mice were randomized and treated either untreated (PBS control) or with a single intravenous administration of 1 × 10⁵ TCID50 particles. Tumor volume (B) and survival rate (C) were then recorded. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5] Schematic diagrams of typical recombinant VSVs according to several embodiments. VSV-hIFNβ-NIS: VSV Indiana was manipulated to express human interferon-β (hIFNβ) in the M / G intergenic region and human sodium iodide symporter (NIS) in the G / L intergenic region, and rescued as described elsewhere (Naik et al., Leukemia, 26:1870-78 (2012)). VSVs expressing CDV-F22458 / 16 (where the signal peptide of the CDV F polypeptide is replaced by the signal peptide of the MeV F polypeptide) and CDV-H5804 were generated using the pVSV-Smart Platform. Point mutations Y539A and R529A in the CDV-H5804 polypeptide were introduced by site-directed mutagenesis to remove native tropism to canine Nectin-4 and SLAMF1 polypeptides, respectively. Targeted viruses were generated by displaying an EGFR or CD38-targeted scFv at the C-terminus of CDV-H5804 with an IGES linker peptide and an H6 polyhistidine tag (SEQ ID NO: 21). The retargeted VSV-CDV F / H constructs were rescued on Vero anti-H6, enabling infection, viral amplification, and fusion with target cells. The titers of each recombinant virus are shown. [Figure 6]Monolayers of specified CHO cells (wild-type CHO cells or CHO cells stably overexpressing the specified receptor) were mock-infected or infected with VSV-CDVFH-GFP or VSV-CDVF / Haa-αEGFR-GFP (MOI = 0.2). Fluorescence micrographs were taken at 100x magnification at the indicated time. GFP expression (green) correlates with viral infection and spread within the monolayer. [Figure 7] A monolayer of specified CHO cells (wild-type CHO cells or CHO cells stably expressing the receptor EGFR or CD38) was infected with VSV-CDVFHaa-αEGFR or VSV-CDVFHaa-αCD38 (MOI = 0.1). After 42 hours, the cell monolayer was fixed with paraformaldehyde and stained with crystal violet. Photographs were taken at 40x magnification. [Figure 8] Therapeutic efficacy of chimeric VSV-CDVFHaa-αEGFR for xenografted human ovarian cancer in the peritoneum. Female 5-6 week old nude mice with thymus defects (Envigo, Indianapolis, IN) were intraperitoneally transplanted with 2 × 10⁶ SKOV3ip.1-Fluc cells (200 μL / mouse) (-7 days). On post-transplant day 7 (day 0), tumor-bearing mice were randomized based on firefly luciferase signaling using IVIS spectroscopy (Perkin Elmer, Hopkinton, MA). Mice were identified by microchip and ear notch. After randomization, mice received a single intraperitoneal injection of either 1 × 10⁷ TCID50 virus or saline control (250 μL / mouse). Mice were euthanized if they developed subcutaneous injection site tumors with ascites exceeding 10% of body weight, or if their body weight decreased by more than 20%. All surviving mice were euthanized at the end of the experiment (92 days after viral treatment). Kaplan-Meier survival curves were plotted and compared using log-rank tests. Clinical observations and body weight were recorded three times a week until the end of the study or euthanasia of the mice. [Figure 9-1]Figure 9 shows the nucleic acid sequence (SEQ ID NO: 1) of the CDV H open reading frame encoding the CDV H polypeptide (SEQ ID NO: 2). [Figure 9-2] This is a continuation of Figure 9-1. [Figure 10-1] Figure 10 shows the nucleic acid sequence (SEQ ID NO: 3) of the CDV F open reading frame encoding the CDV F polypeptide (SEQ ID NO: 4). [Figure 10-2] This is a continuation of Figure 10-1. [Figure 11-1]Retargeting of the wild-type CDV envelope to EGFR and CD38. (A) Schematic diagram of the cloning strategy for generating the retargeted wild-type CDV H polypeptide (top). Standard single-letter amino acid abbreviations are used to indicate the changes introduced to eliminate the use of innate receptors (SLAMF1 and Nectin-4) (bottom). Amino acid numbering is based on SEQ ID NO: 5. Single-chain antibody fragments are displayed as C-terminal extensions of the H glycoprotein using the Factor Xa (Fxa) cleavage site (IEGR amino acid sequence). A 6-histidine tag (SEQ ID NO: 21) is included in all constructs as needed to facilitate viral rescue on Vero-His cells. (B) Simultaneous transfection experiments demonstrating the targeting fusion capability of the CD38-targeted receptor-blind H polypeptide. CHO-CD38 cells in 12-well plates were simultaneously transfected with either a CMV-driven CDV F plasmid and CMV-driven wild-type CDV H-CD38 or CMV-driven receptor-blind CDV H-CD38. Cells were fixed, stained, and imaged after 24 hours. (C) Targeted cell fusion by the CDV H construct was resistant to pooled measles-immunized human serum. CHO-CD38 cells were simultaneously transfected with CMV-driven H and F plasmids, along with a CMV-driven GFP plasmid for visualization, and incubated with the indicated dilutions. Cells were photographed 24 hours after transfection. (D) Chimeric measles viruses with targeted CDV H polypeptides retain specificity to CHO cells expressing the target receptor and to a panel of human tumor cell lines with the target receptor. Cell lines were infected with each virus at a MOI of 0.5 and photographed after 48 hours. (E) Schematic design of an experiment to verify the in vivo oncolytic effect and specificity of chimeric measles virus with a retargeted CDV envelope. Nude mice were transplanted with 5 × 10⁶ SKOV3 ip-fluc cells subcutaneously (SQ) or intraperitoneally (IP). Starting on day 10, 1 × 10⁶ TCID 50 / mL was administered intratumorally (IT) to subcutaneous tumors and 2 × 10⁶ TCID 50 / mL in IP tumors, every other day for 6 doses.(F) Individual tumor volume of SKOV3ip subcutaneous tumors treated with each virus (top) and survival rate of animals with intraperitoneal tumors treated with each virus (bottom). [Figure 11-2] This is a continuation of Figure 11-1. [Figure 11-3] This is a continuation of Figure 11-2. [Figure 12-1] Figure 12 shows alignments of a representative number of CDV H polypeptides. The top row sequence (referred to as AF378705.1_America1) is sequence number 5, which is used for numbering at the indicated location. Figure 12 discloses sequence numbers 5, 1-109, 105, 110-145, and 227-228 in that order. [Figure 12-2] This is a continuation of Figure 12-1. [Figure 12-3] This is a continuation of Figure 12-2. [Figure 12-4] This is a continuation of Figure 12-3. [Figure 12-5] This is a continuation of Figure 12-4. [Figure 12-6] This is a continuation of Figure 12-5. [Figure 12-7] This is a continuation of Figure 12-6. [Figure 12-8] This is a continuation of Figure 12-7. [Figure 12-9] This is a continuation of Figure 12-8. [Figure 12-10] This is a continuation of Figure 12-9. [Figure 12-11] This is a continuation of Figure 12-10. [Figure 12-12] This is a continuation of Figure 12-11. [Figure 12-13] This is a continuation of Figure 12-12. [Figure 12-14] This is a continuation of Figure 12-13. [Figure 12-15] This is a continuation of Figure 12-14. [Figure 12-16] This is a continuation of Figure 12-15. [Figure 13-1]Figure 13 shows alignments of a representative number of CDV F polypeptides. The signal peptide sequence is from amino acid position 1 to amino acid position 135. The top row sequence (referred to as AF378705.1_America1) is sequence number 7, which is used for numbering at the indicated location. Figure 13 discloses sequence numbers 7 and 146-226 in this order. [Figure 13-2] This is a continuation of Figure 13-1. [Figure 13-3] This is a continuation of Figure 13-2. [Figure 13-4] This is a continuation of Figure 13-3. [Figure 13-5] This is a continuation of Figure 13-4. [Figure 13-6] This is a continuation of Figure 13-5. [Figure 13-7] This is a continuation of Figure 13-6. [Figure 13-8] This is a continuation of Figure 13-7. [Figure 13-9] This is a continuation of Figure 13-8. [Figure 13-10] This is a continuation of Figure 13-9. [Figure 13-11] This is a continuation of Figure 13-10. [Figure 14] CDV OL can infect cells lacking SLAMF1 and NECTIN4 receptors. (A) The infectivity of CDV isolates to Vero cells was evaluated compared to OL strains. Cells were infected with an MOI of 0.1 (determined by Vero-dog SLAMF1 cells) and stained with Hema-Quick for visualization after 48 hours. (B) A panel of CHO cells expressing different related receptors was infected with eGFP reporter MeV containing CDV H / F OL glycoprotein. Infectivity was reported using fluorescence microscopy. Magnification: 40x. [Figure 15-1]The heterogeneous combination of wild-type CDV H / F and the truncated signal peptide promotes receptor-dependent fusion due to weakened HF interaction. (A) Syncytium formation in cells simultaneously transfected with CDV-F, CDV-H, and eGFP. The signal peptide of CDV-F was replaced with a homolog of MeV-F, as shown in the black box in the schematic diagram. Fusion scores were assessed under the GFP channel 24 hours after simultaneous transfection. (B) Quantitative fusion assay. Effector BHK cells were transfected with one side of the dual-split reporter plasmid in addition to the indicated combination of adherent protein (CDV-H or Nipah-G) and fusion protein (F). Target CHO cells and CD38-expressing CHO cells (CHO-CD38) were transfected with the other side of the dual-split reporter plasmid. Cells were overlaid 16 hours after transfection, and sea urchin luciferase activity was measured at 8 hours (RLU). The values ​​represent the mean ± standard deviation (SD) of one representative experiment performed in a triple series. Statistical significance was determined using one-way ANOVA with Holm-Sidak multiple comparison test (ns, not significant; *, p<0.05; **, p<0.002; ***, p<0.0001). (C) Immunoprecipitation of CDV-H / F. HEK293T cells transiently expressing either wt or mutant HIS-tagged CDV-H protein together with FLAG-tagged CDV-F protein were lysed and immunoprecipitated (IP) with anti-FLAG antibody. Signal intensity was measured using anti-HIS antibody. (D) Quantitative fusion assay of fully retargeted CDV-H and MeV-H proteins into CHO cells and CHO cell-derived cell lines. MeV-H / F and CDV-H / F complexes, either HIS-tagged or HIS-tagged and retargeted to CD38, were transfected into effector cells, and luminescence signals were measured over time. MeV-Haals = MeV-H unavailable for CD46, NECTIN-4, and SLAMF1 via mutations Y481A, R533A, S548L, and F549S (blind). [Figure 15-2]This is a continuation of Figure 15-1. [Figure 15-3] This is a continuation of Figure 15-2. [Figure 15-4] This is a continuation of Figure 15-3. [Figure 16-1] Conservation of amino acid residue M437 of the CDV-H protein across different gene groups. Sequence alignment was performed using CDV-H sequences retrieved from GenBank, which include the CDV-H sequence determined in this study for the SPA.Madrid / 22458 / 16 isolate. Accession numbers are shown. [Figure 16-2] This is a continuation of Figure 16-1. [Figure 16-3] This is a continuation of Figure 16-2. [Figure 16-4] This is a continuation of Figure 16-3. [Figure 17-1] Integrity and expression of receptor-binding proteins displaying chimeric ligands. (A) Western blot analysis of HEK293T cells transfected with a specified protein fused to anti-CD38 scFv or an unfused specified protein. Proteins were blotted with anti-HIS antibody or anti-β-actin antibody (loading control). (B) Flow cytometry analysis of adherent and mutant protein expression on fixed HEK293T cells with or without permeabilization. Histograms are obtained from one representative experiment from two repeated biological experiments. The geometric mean intensity ± SD from two repeated biological experiments is shown in the upper right corner of each histogram. The filled curve region indicates cells transfected with an empty plasmid. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18-1]Insertion of a FLAG tag into the F ectodomain and its effect on the bioreactivity of the protein. (A) Schematic diagrams of uncleaved MeV-F and CDV-F. The NH2 and COOH ends, signal peptide (SP), fusion peptide (FP), and transmembrane (TM) and cytoplasmic regions are shown. Sequences around the cleavage site (bold) and the fusion peptide sequence are shown. Numbering considers isomorphic signal peptides. (B) Syncytium formation in Vero cells after simultaneous transfection with homologous H and F expression plasmids having FLAG insertions at different positions. Cells were stained 16 hours after transfection and micrographs were taken for quantification. (C) Quantification of syncytium formation. Data are shown as mean ± SD (n=20). Significance was determined using one-way ANOVA with Holm-Sidak multiple comparison test (ns, not significant; ***, p≦0.001). (D) Dual-split protein fusion assay for simultaneous transfection of CDV-H / F SPA with or without FLAG tag insertion at amino acid 216. Luciferase signal was measured after 8 hours. Experiments were technically performed in two sets. [Figure 18-2] This is a continuation of Figure 18-1. [Figure 19] CD46 specificity of scFv. (A) SDS-PAGE analysis of target protein. MW: molecular weight ladder, C: Coomassie staining, WB: Western blot analysis using anti-CD46 antibody. (B) Size exclusion chromatography trace of CD46 used in the experiment. Estimated MW is obtained from the calibration curve as shown in the figure. (C) Binding of scFv-Fc-tagged fusion protein to CD46 or NECTIN4 as measured by ELISA. Detection was performed using the Fc portion as a protein amount control. Experiments were technically performed in pairs. Data are shown as mean ± SD (n=2). Significance was determined using one-way ANOVA with Holm-Sidak multiple comparison test. *, p<0.05; **, p<0.005. [Figure 20-1]The displayed CD46 binding affinity of scFv determines the CD46-dependent intercellular fusion of the retargeted CDV H / F complex. (A) Representative sensorgrams (resonance units, RU) regarding the binding of CD46 to biosensor surfaces containing (solid line) or not containing (dashed line) single-chain antibody fragment (scFv). Experimental data represent the injection of scFv K2 for 300 seconds, followed by the injection of buffer. Subsequently, 1 μM CD46 was flowed over both biosensor surfaces, and signals were recorded during injection (binding) and after injection (dissociation). The surfaces were finally regenerated at the end of the cycle as described herein. (B) Binding of CD46 to scFv as assessed by surface plasmon resonance. Sensorgrams showing response units (black lines) to scFv at various concentrations of CD46. The optimal 1:1 binding model is shown as a red dashed line. Binding affinity (Kd) was determined from the binding and dissociation rates (Table 1). (C) Quantitative fusion assay of MeV-H and CDV-H variants on CHO cells. The experiment was performed in a double-row setup and repeated twice, yielding similar results (see Figure 21). Data are shown as mean ± SD. [Figure 20-2] This is a continuation of Figure 20-1. [Figure 21] The binding affinity of scFv displayed on the CDV-H / F complex promotes cell-cell fusion. (A) Cell enzyme-linked immunosorbent assay (CELISA) for the amount of cellular protein used in the quantitative fusion assay in Figure 20C. CELISA was performed on CHO cells transfected with the indicated adherent protein using anti-6×HIS-tag monoclonal antibody (n=5). (B) Quantitative fusion assay of CD46 retargeted CDV-H / F complex using affinity-adjusted scFv (same data as presented in Figure 20C). Y539A indicates substitution in CDV-H to eliminate native tropism against NECTIN4. [Figure 22-1]CD46-retargeted CDV envelope glycoproteins are determinant of tropism. (A) Schematic diagram of Stealth: Vaccine-derived measles virus pseudotyped with CD46-retargeted CDV H / F envelope protein. Created with BioRender.com. (B) Role of CD46 binding affinity in viral entry. Cells were infected with Stealth virus displaying scFv with different affinities to CD46 at specified MOIs. eGFP expression was monitored 48 hours after infection. (C) CHO cells and derived cell lines expressing HIS-pseudoreceptors or CD46 were infected with Fluc-expressing Stealth virus (K1 and A09) at MOI 0.5. Luciferase expression was measured 48 hours after infection. n=2, *, p<0.05 (two-sided t-test) except for CHO-CD46. (D) Multistep proliferation dynamics of Stealth-A09 in Vero or Vero-αHIS cells. At the indicated time, both the supernatant and cell pellet were collected, and viral titers were measured in Vero-αHIS cells. Values ​​and error bars (SD) were determined for representative experiments performed in triplicates. (E) Protein composition of the virus. Western blot analysis was performed using equal amounts of virus particles, with the relevant antibody used as a probe. Molecular weight of the standard substance is shown. (F) Viral tropism. CHO cell-derived cell lines were infected with the specified eGFP-expressing virus. eGFP autofluorescence was measured after 48 hours. Scale bar, 200 μm. (G) Genetic stability of Stealth. Vero-hSLAMF1 cells were infected with Stealth and passaged multiple times. After 8 passages, the collected virus was used to infect human or canine SLAMF1-expressing Vero cells. Representative micrographs after 3 or 6 days of infection are shown. [Figure 22-2] This is a continuation of Figure 22-1. [Figure 22-3] This is a continuation of Figure 22-2. [Figure 22-4] This is a continuation of Figure 22-3. [Figure 23]Evaluation of receptor interactions with modified CDV fusion mechanism (apparatus) complexes. Cells were co-transfected with MeV-F and MeV-H, or CDV-F and CDV-H retargeting variants, along with CD46-specific scFv. For visualization purposes, expression plasmids encoding eGFP were co-transfected, and the autofluorescence of eGFP was visualized 24 hours after transfection. Y539A indicates substitution in CDV-H to eliminate native tropism against NECTIN4. The "+" and "-" symbols were used for semi-quantification (same as shown in Figure 15A). "No display" indicates the absence of scFv. [Figure 24-1] High CD46 binding affinity determines the oncolytic activity of CD46-targeted Stealth virus in a mouse model of ovarian cancer. (A) Schematic diagram of the experimental design. SKVOv3ip.1 tumor cells (SKOV3ip.Fluc) encoding the firefly luciferase gene were intraperitoneally transplanted into thymus-deficient mice. On day 10, 1 × 10⁶ TCID50 particles of Stealth were administered via the same route. Subsequently, tumor tissue volume was monitored at 7-day intervals by bioluminescence imaging (BLI). (B) Kaplan survival curves (n=5) of SKOV3ip.Fluc-bearing mice treated with Stealth-N1E and Stealth-A09 viruses. Statistical significance is defined by the log-rank test. (C) Representative BLI showing dorsal imaging of treated animals. Radiance (photons / sec / cm / steradian, p / s / cm² / sr) was converted to color to indicate the tumor tissue volume of the mice, as explained on the right. (D) Quantification of whole-body luminescence (photons / second / cm / steradian, p / s / cm² / sr). n=5. Ns, not significant; *, p<0.05; **, p<0.005. [Figure 24-2] This is a continuation of Figure 24-1. [Figure 25]Stealth-A09 virus achieves oncolytic activity indistinguishable from that of the parent virus, MeV, in a mouse model of multiple myeloma. (A) SCID mice with subcutaneous U266.B1 cell tumors were intravenously treated with suboptimal doses of the virus. Tumor growth was measured with calipers (n=5), and animals were euthanized when the tumor ulcerated or when the tumor size reached 20% of body weight. (B) Kaplan-Meier survival curves (n=5). Significant differences between groups were determined by the log-rank test (*, p<0.05). (C) Viral transport to subcutaneous tumor cells after systemic administration. eGFP expression was evaluated by immunohistochemistry in two representative samples from each group collected at the time of euthanasia. Scale, 200 nm. [Figure 26] Increased binding affinity to CD46 promotes CD46-specific viral entry. Fluc-expressing Stealth viruses were used to infect the target cells with reduced MOI. Luciferase expression was measured 48 hours after infection. n=2 was used for all except CHO-CD46 and Stealth-A09 (n=3). [Figure 27-1] Stealth virus retains oncolytic properties in the presence of MeV-immunized serum. (A) SKOV3ip.Fluc cells were injected into thymus-deficient nude mice and immobilized for 10 days. Then, 600 mIU of anti-MeV IgG antibody was administered intraperitoneally to the mice in the relevant group 3 hours before viral treatment via the same route. (B) Kaplan-Meier survival curves (n=5 mice per group). Significant differences between groups were determined by the log-rank test (ns, not significant; *, p<0.05; **, p<0.002). (C) Representative BLI showing dorsal imaging of treated animals. Radiance (photons / sec / cm / steradian, p / s / cm2 / sr) was converted to color to indicate the amount of tumor tissue in the mice, as explained on the right. (D) Quantification of whole-body luminescence (photons / sec / cm / steradian, p / s / cm2 / sr). n=5. Statistical significance was determined by one-way ANOVA using Dunnett's multiple comparison test. Ns, not significant. [Figure 27-2] This is a continuation of Figure 27-1. [Figure 27-3]This is a continuation of Figure 27-2. [Figure 28] Lack of cross-neutralization between measles virus and Stealth. (A) Viral neutralization assays of MeV and Stealth. Human AB pooled serum (left panel) or ferret anti-CDV serum (right panel) were used. Relative infection refers to the infectious dose in the presence of serum compared to the infectious dose in the absence of serum. Values ​​were calculated from two or three biological replicate experiments, which were technically performed in four-sequences. (B) Antisera obtained from infected HuCD46Ge-IFNARKO mice was also used to determine cross-neutralization between viruses, n=8 (note that some data points overlap). ND50 titers were converted to mIU / mL based on the ND50 obtained for MeV when evaluated against the Third WHO International Serological Standard (3 IU / mL). [Modes for carrying out the invention]

[0030] Detailed explanation This specification provides CDV F polypeptides. As described herein, CDV F polypeptides can be designed so that viral particles containing CDV F polypeptides, together with CDV H polypeptides, exhibit high fusion activity. For example, CDV F polypeptides can be designed to contain a signal peptide sequence of 75 amino acids or less in length. Typically, wild-type CDV F polypeptides contain a signal peptide sequence of approximately 135 amino acids in length. An example of a 135-amino acid signal peptide sequence of wild-type CDV F polypeptide is SEQ ID NO: 6(MHKEIPEKSRTRTHTQQDLPQQKSTEYTEIKTSRARHGITPAQRSTHYGPRTLDRLVCYIMNRAMSCKQASYRSDNIP As shown in AHGDHEGVVHHTPESVSQGARSQLKRRTSNAINSGFQYIWLVLWCIGIASLFLCSKA), truncating the signal peptide sequence of the CDV F polypeptide to a length of 75 amino acids or less, as described herein, can result in a CDV F polypeptide that, when part of the virus together with the CDV H polypeptide, enables increased viral fusion activity compared to the fusion activity levels shown by an equivalent control virus containing the CDV F polypeptide having the full-length wild-type signal peptide sequence (e.g., SEQ ID NO: 6).

[0031] The CDV F polypeptides described herein may contain signal peptide sequences having a length of 7 to 75 amino acids. For example, the CDV F polypeptides described herein may contain signal peptide sequences having a length of 7 to 75 amino acids (e.g., 7 to 70, 7 to 65, 7 to 60, 7 to 55, 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, 10 to 75, 15 to 75, 20 to 75, 25 to 75, 35 to 75, 45 to 75, 50 to 75, 55 to 75, 65 to 75, 20 to 60, 25 to 50, 30 to 60, or 30 to 40). The CDV F polypeptides described herein can be prepared by truncating a wild-type signal peptide sequence from its N-terminus, from its C-terminus, or from both its N-terminus and C-terminus, or by deleting amino acids from the region between the N-terminus and C-terminus of the wild-type signal peptide sequence. In some cases, measles virus signal peptide sequences can be used as signal peptides for the CDV F polypeptides described herein. Examples of signal peptide sequences for the CDV F polypeptides described herein include, but are not limited to, those listed in Table 1.

[0032] [Table 1]

[0033] In some cases, the CDV F polypeptides described herein can be designed to lack the entire signal peptide sequence. For example, the CDV F polypeptides described herein may have one of the amino acid sequences shown in Figure 13, beginning with the 140th amino acid.

[0034] The CDV F polypeptides described herein may have any suitable amino acid sequence, as long as the CDV F polypeptide does not contain a signal peptide sequence longer than 75 amino acid residues. Examples of amino acid sequences of CDV F polypeptides that can be used as described herein include, but are not limited to, the amino acid sequences shown in Figure 13.

[0035] Furthermore, this specification provides CDV H polypeptides. As described herein, CDV H polypeptides can be designed such that viruses containing the CDV H polypeptide together with the CDV F polypeptide exhibit reduced or eliminated tropism against SLAMF1 polypeptide and / or Nectin-4 polypeptide compared to viruses containing the wild-type CDV H polypeptide. For example, CDV H polypeptides can be designed to contain mutations at one or more amino acid positions among 454, 460, 479, 494, 510, 520, 525, 526, 527, 528, 529, 537, 539, 547, and 548 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). Normally, viruses containing the wild-type CDV H polypeptide (along with the CDV F polypeptide) exhibit tropism to the SLAMF1 and Nectin-4 polypeptides, such that the virus infects SLAMF1-positive and Nectin-4-positive cells. As described herein, the ability of a virus containing the CDV H polypeptide (along with the CDV F polypeptide) to infect SLAMF1-positive and / or Nectin-4-positive cells can be reduced or eliminated by mutating one or more of the amino acid positions P / S454, V / L / F460, L / F / W479, I494, I / L / V510, Y520, Y / N525, D / G526, I / V527, S / T528, R529, Y / D537, Y539, Y / F547, and Y / M548 of the CDV H polypeptide to a different amino acid (e.g., alanine).Examples of CDV H polypeptides described herein that exhibit reduced or eliminated tropism against SLAMF1 polypeptide and / or Nectin-4 polypeptide include, but are not limited to, the CDV H polypeptides shown in Figure 12, provided that the CDV H polypeptide contains one or more mutations from P / S454, V / L / F460, L / F / W479, I494, I / L / V510, Y520, Y / N525, D / G526, I / V527, S / T528, R529, Y / D537, Y539, Y / F547, and Y / M548 (for example, 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations). Examples of mutations that can be used to produce CDV H polypeptides with reduced or eliminated tropism against the SLAMF1 polypeptide and / or Nectin-4 polypeptide are, but are not limited to, those listed in Table 2. Examples of combinations of the mutations listed in Table 2 that can be used to produce CDV H polypeptides with reduced or eliminated tropism against the SLAMF1 polypeptide and / or Nectin-4 polypeptide are, but are not limited to those listed in Table 3.

[0036] [Table 2]

[0037] [Table 3]

[0038] This specification also provides recombinant viruses (e.g., VSV) containing the CDV H polypeptide and / or the CDV F polypeptide described herein, and also provides methods for producing recombinant viruses (e.g., VSV) containing the CDV H polypeptide and / or the CDV F polypeptide described herein. For example, a recombinant virus (e.g., VSV) can be designed to (a) contain the CDV H polypeptide and wild-type CDV F polypeptide described herein, (b) contain the wild-type CDV H polypeptide and the CDV F polypeptide described herein, or (c) contain the CDV H polypeptide and the CDV F polypeptide described herein. In some cases, a recombinant virus (e.g., VSV) can be designed to contain the CDV H polypeptide having CDV H 5804 and the CDV F polypeptide having CDV F 22458 / 16.

[0039] This specification also provides nucleic acid molecules encoding the CDV H polypeptide described herein and / or the CDV F polypeptide described herein. For example, a nucleic acid molecule (e.g., a vector) can be designed to encode the CDV H polypeptide described herein and / or the CDV F polypeptide described herein.

[0040] This specification provides methods and materials related to VSV. For example, this specification provides replicable VSV, nucleic acid molecules encoding replicable VSV, methods for producing replicable VSV, and methods for using replicable VSV for the treatment of cancer or infectious diseases.

[0041] As described herein, VSV can be designed to have nucleic acid molecules that encode VSV N polypeptide, VSV P polypeptide, VSV M polypeptide, CDV F polypeptide (e.g., the CDV F polypeptide described herein), CDV H polypeptide (e.g., the CDV H polypeptide described herein), and VSV L polypeptide, but not functional VSV G polypeptide. Naturally, the sequences described herein for VSV are incorporated into plasmids that encode the positive-strand cDNA of the viral genome, enabling the generation of the VSV minus-strand genome. Thus, a nucleic acid sequence encoding a VSV polypeptide, for example, is naturally considered to refer to an RNA sequence that serves as a template for the positive-strand transcript encoding that polypeptide (e.g., via direct translation).

[0042] The nucleic acids encoding the CDV F polypeptide and the CDV H polypeptide can be placed at any position within the VSV genome. In some cases, the nucleic acids encoding the CDV F polypeptide and the CDV H polypeptide can be placed downstream of the nucleic acid encoding the VSV M polypeptide. For example, the nucleic acids encoding the CDV F polypeptide and the CDV H polypeptide can be placed between the nucleic acid encoding the VSV M polypeptide and the nucleic acid encoding the VSV L polypeptide.

[0043] Any suitable nucleic acid encoding the CDV F polypeptide can be inserted into the VSV genome. For example, the nucleic acid encoding the wild-type CDV F polypeptide or the CDV F polypeptide described herein can be inserted into the VSV genome.

[0044] Any suitable nucleic acid encoding the CDV H polypeptide can be inserted into the VSV genome. For example, nucleic acids encoding the wild-type H polypeptide or the H polypeptide described herein can be inserted into the VSV genome. In some cases, nucleic acids encoding the CDV H polypeptide lacking specificity for SLAMF1 and / or Nectin-4 can be inserted into the VSV genome. For example, nucleic acids encoding the CDV H polypeptide having one or more mutations as shown in Table 2 can be inserted into the VSV genome. In some cases, the VSV / CDV hybrids described herein can be designed to have a pre-selected tropism. For example, using CDV F and / or H polypeptides with knocked-out specificity for SLAMF1 and / or Nectin-4, scFv or polypeptide ligands can be bound, for example, to the C-terminus of the CDV H polypeptide. In such cases, scFv or polypeptide ligands can determine the tropism of the VSV / CDV hybrid. Examples of scFvs that can be used to direct VSV / CDV hybrids to cell receptors (e.g., tumor-associated cell receptors) include, but are not limited to, anti-EGFR, anti-CD46, anti-αFR, anti-PSMA, anti-HER-2, anti-CD19, anti-CD20, or anti-CD38 scFvs. Examples of polypeptide ligands that can be used to direct VSV / CDV hybrids include, but are not limited to, urokinase-type plasminogen activator uPA polypeptide, cytokines such as IL-13, single-chain T cell receptor (scTCR), exstatin polypeptide, and integrin-binding polypeptide.

[0045] In some cases, the nucleic acid molecules of VSV described herein may encode IFN polypeptides, fluorescent polypeptides (e.g., GFP polypeptides), NIS polypeptides, therapeutic polypeptides, innate immune antagonist polypeptides, tumor antigens, or combinations thereof. A nucleic acid encoding an IFN polypeptide may be positioned downstream of a nucleic acid encoding a VSV M polypeptide. For example, a nucleic acid encoding an IFN polypeptide may be positioned between a nucleic acid encoding a VSV M polypeptide and a nucleic acid encoding a CDV F polypeptide or a CDV H polypeptide. Such a configuration may allow the virus to express an effective amount of IFN polypeptide to activate an antiviral innate immune response in non-cancerous tissues without interfering with efficient viral replication in cancer cells, thus mitigating potential virogenicity.

[0046] Any suitable nucleic acid encoding an IFN polypeptide can be inserted into the VSV genome. For example, a nucleic acid encoding an IFNβ polypeptide can be inserted into the VSV genome. Examples of nucleic acids encoding IFNβ polypeptides that can be inserted into the VSV genome include, but are not limited to, the nucleic acid encoding the human IFNβ polypeptide with the nucleic acid sequence described in GenBank® accession number NM_002176.2 (GI No. 50593016), the nucleic acid encoding the mouse IFNβ polypeptide with the nucleic acid sequences described in GenBank® accession number NM_010510.1 (GI No. 6754303), BC119395.1 (GI No. 111601321), or BC119397.1 (GI No. 111601034), and the nucleic acid encoding the rat IFNβ polypeptide with the nucleic acid sequence described in GenBank® accession number NM_019127.1 (GI No. 9506800).

[0047] The nucleic acid encoding the NIS polypeptide can be positioned downstream of the nucleic acid encoding the CDV F polypeptide or the nucleic acid encoding the CDV H polypeptide. For example, the nucleic acid encoding the NIS polypeptide can be positioned between the nucleic acid encoding the CDV F or H polypeptide and the nucleic acid encoding the VSV L polypeptide. Such a configuration allows the virus to express an amount of NIS polypeptide that is effective in (a) enabling selective accumulation of iodine in infected cells, but (b) not high enough to be toxic to infected cells, thereby enabling both imaging of the viral distribution using radioisotopes and radiotherapy targeting infected cancer cells.

[0048] Any suitable nucleic acid encoding a NIS polypeptide can be inserted into the VSV genome. For example, a nucleic acid encoding a human NIS polypeptide can be inserted into the VSV genome. Examples of nucleic acids encoding NIS polypeptides that can be inserted into the VSV genome include nucleic acids encoding human NIS polypeptides with nucleic acid sequences described in GenBank® accession numbers NM_000453.2 (GI No. 164663746), BC105049.1 (GI No. 85397913), or BC105047.1 (GI No. 85397519), nucleic acids encoding mouse NIS polypeptides with nucleic acid sequences described in GenBank® accession numbers NM_053248.2 (GI No. 162138896), AF380353.1 (GI No. 14290144), or AF235001.1 (GI No. 12642413), and GenBank® accession number XM_524154 (GI Examples include, but are not limited to, nucleic acids encoding chimpanzee NIS polypeptide with the nucleic acid sequence described in No. 114676080, nucleic acids encoding canine NIS polypeptide with the nucleic acid sequence described in GenBank® accession number XM_541946 (GI No. 73986161), nucleic acids encoding bovine NIS polypeptide with the nucleic acid sequence described in GenBank® accession number XM_581578 (GI No. 297466916), nucleic acids encoding porcine NIS polypeptide with the nucleic acid sequence described in GenBank® accession number NM_214410 (GI No. 47523871), and nucleic acids encoding rat NIS polypeptide with the nucleic acid sequence described in GenBank® accession number NM_052983 (GI No. 158138504).

[0049] The VSV nucleic acid sequences described herein, which encode the VSV N polypeptide, VSV P polypeptide, VSV M polypeptide, and VSV L polypeptide, may be derived from the VSV Indiana strain described in GenBank® accession number NC_001560 (GI No. 9627229) or from the VSV New Jersey strain.

[0050] In one embodiment, this specification provides a VSV containing a nucleic acid molecule (e.g., an RNA molecule) having (for example, in the 3' to 5' direction) nucleic acid sequences that serve as templates for a positive-strand transcript encoding a VSV N polypeptide, a VSV P polypeptide, a VSV M polypeptide, a CDV F polypeptide, a CDV H polypeptide, and a VSV L polypeptide, but lacking a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a functional VSV G polypeptide. Such a VSV may infect cells (e.g., cancer cells) and may be able to replicate.

[0051] Nucleic acids (e.g., nucleic acids encoding CDV F polypeptide, CDV H polypeptide, IFN polypeptide, and / or NIS polypeptide) can be inserted into the genome of VSVs using any suitable method. For example, nucleic acids can be inserted into the genome of VSVs using methods described in other literature (Schnell et. al., PNAS, 93:11359-11365 (1996), Obuchi et al., J. Virol., 77(16):8843-56 (2003)); Goel et al., Blood, 110(7):2342-50 (2007)); and Kelly et al., J. Virol., 84(3):1550-62 (2010)). VSVs containing the nucleic acid molecules described herein can be identified using any suitable method. Such methods include, but are not limited to, PCR, as well as nucleic acid hybridization techniques such as Northern and Southern spectroscopy. In some cases, immunohistochemical and biochemical techniques can be used to detect the expression of polypeptides encoded by specific nucleic acid molecules, thereby determining whether a VSV contains a particular nucleic acid molecule.

[0052] In another embodiment, this specification provides nucleic acid molecules that encode VSV N polypeptide, VSV P polypeptide, VSV M polypeptide, CDV F polypeptide, CDV H polypeptide, and VSV L polypeptide, but lack the ability to encode a functional VSV G polypeptide. For example, the nucleic acid molecule provided herein may be a single nucleic acid molecule that includes nucleic acid sequences encoding a VSV N polypeptide, a nucleic acid sequence encoding a VSV P polypeptide, a nucleic acid sequence encoding a VSV M polypeptide, a nucleic acid sequence encoding a CDV F polypeptide, a nucleic acid sequence encoding a CDV H polypeptide, and a nucleic acid sequence encoding a VSV L polypeptide, but does not include a nucleic acid sequence encoding a functional VSV G polypeptide.

[0053] In another aspect, this specification provides nucleic acid molecules that lack the ability to encode a functional VSV G polypeptide but encode VSV N polypeptide, VSV P polypeptide, VSV M polypeptide, IFN polypeptide, CDV F polypeptide, CDV H polypeptide, NIS polypeptide, and VSV L polypeptide. For example, the nucleic acid molecules provided herein may be a single nucleic acid molecule comprising nucleic acid sequences encoding a VSV N polypeptide, a nucleic acid sequence encoding a VSV P polypeptide, a nucleic acid sequence encoding a VSV M polypeptide, a nucleic acid sequence encoding an IFN polypeptide, a nucleic acid sequence encoding a CDV F polypeptide, a nucleic acid sequence encoding a CDV H polypeptide, a nucleic acid sequence encoding an NIS polypeptide, and a nucleic acid sequence encoding a VSV L polypeptide, but lacking the ability to encode a functional VSV G polypeptide.

[0054] As used herein, the term “nucleic acid” encompasses both RNA (e.g., viral RNA) and DNA (cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA). Nucleic acids may be double-stranded or single-stranded. Single-stranded nucleic acids may be sense strands or antisense strands. Nucleic acids may also be circular or linear.

[0055] This specification also provides methods for treating cancer (e.g., reducing tumor size, inhibiting tumor growth, or decreasing the number of surviving tumor cells), methods for inducing host immunity against cancer, and methods for treating infectious diseases such as HIV or measles. For example, recombinant viruses (e.g., VSV) provided herein can be administered to mammals with cancer to reduce tumor size, inhibit the growth of cancer cells or tumors, decrease the number of surviving cancer cells in the mammalian body, and / or induce a host immune response against the tumor. Recombinant viruses (e.g., VSV) provided herein can be grown in host cells to increase the number of available copies of the virus typically by at least twofold (e.g., 5-10fold, 50-100fold, 500-1,000fold, or as many as 5,000-10,000fold). In some cases, recombinant viruses (e.g., VSV) provided herein can be grown in standard cell culture media (e.g., 5% CO2 at 37°C). 2 The virus can be grown in DMEM or RPMI-1640 (containing 5-10% fetal bovine serum) until the desired concentration is achieved. Viral titer is typically assessed by inoculating cultured cells (e.g., Vero cells).

[0056] The recombinant viruses (e.g., VSV) provided herein can be administered to cancer patients, for example, by direct injection into cancer cell populations (e.g., tumors) or by intravenous administration to cancer cells. The recombinant viruses (e.g., VSV) provided herein can be used to treat various types of cancer, including, but not limited to, myeloma (e.g., multiple myeloma), melanoma, glioma, lymphoma, mesothelioma, and cancers of the lung, brain, stomach, colon, rectum, kidney, prostate, ovary, breast, pancreas, liver, and head and neck.

[0057] The recombinant viruses provided herein (e.g., VSV) can be administered to a patient either by direct (e.g., intratumoral) administration into a group of cancer cells or systemic (e.g., intravenous) administration in a biologically compatible solution or a pharmaceutically acceptable delivery vehicle. Suitable pharmaceutical formulations depend in part on the use and route of administration (e.g., transdermal or by injection). Such forms should not prevent the composition or formulation from reaching the target cells (i.e., the cells to which the virus is to be delivered) or from exerting its effect. For example, a pharmacological composition injected into the bloodstream should be soluble.

[0058] The dosage varies depending on the patient (e.g., depending on the size of the tumor), starting from a virus concentration proven to be safe, and while monitoring the reduction of cancer cell growth along with the presence or absence of adverse side effects, increasing gradually to higher doses up to 10 12 pfu, an effective amount can be determined. A therapeutically effective dose typically results in at least a 10% reduction in the number of cancer cells or tumor size. Dose escalation studies can be used to obtain the desired effect for a given viral treatment (see, e.g., Nies and Spielberg, "Principles of Therapeutics," In Goodman & Gilman's The Pharmacological Basis of Therapeutics, eds. Hardman, et al., McGraw-Hill, NY, 1996, pp 43-62).

[0059] The recombinant viruses provided herein (e.g., VSV) are, for example, about 10 3 pfu to about 10 12 pfu (e.g., about 10 5 pfu to about 10 12 pfu, about 10 6 pfu to about 10 11 pfu, or about 10 6 pfu to about 10 10It can be delivered in doses in the range of pfu. A therapeutically effective dose can be provided in repeated doses. Repeated doses are appropriate when either the cancer cell population or tumor has stopped shrinking, or the degree of viral activity is decreasing while the tumor is still present, as indicated by observation of clinical symptoms or tumor size, or by monitoring assays. Repeated doses can be administered via the same route as the initial administration, or a different route. A therapeutically effective dose can be delivered in doses consisting of several distinct parts (e.g., at intervals of several days or several weeks), although in some embodiments, one to about 12 doses are provided. Alternatively, a therapeutically effective dose of recombinant virus (e.g., VSV) provided herein can be delivered by a sustained-release formulation. In some cases, recombinant virus (e.g., VSV) provided herein can be delivered in combination with an agent that promotes viral replication and spread within cancer cells, or an agent that protects non-cancer cells from viral toxicity. Examples of such drugs are described in other literature (Alvarez-Breckenridge et al., Chem. Rev., 109(7):3125-40 (2009)).

[0060] The recombinant viruses (e.g., VSV) provided herein can be administered using a device to provide sustained release. Sustained-release formulations of recombinant viruses (e.g., VSV) provided herein may include, for example, a high molecular weight excipient (e.g., a swellable or non-swellable gel, or collagen). A therapeutically effective dose of the recombinant virus (e.g., VSV) provided herein may be provided within the high molecular weight excipient, which is then embedded in the site of cancer cells (e.g., near or within the tumor). The excipient is gradually dissolved by the action of body fluids, and an effective amount of virus is continuously released over a period of time. Alternatively, the sustained-release device may include a series of alternating active and spacer layers. Each active layer of such a device typically contains a single dose of virus embedded in the excipient, and each spacer layer contains either only the excipient or a low concentration of virus (i.e., below an effective dose). As each successive layer of the device dissolves, the viral dose is delivered as a pulse. The size / formulation of the spacer layer determines the dosing interval, but is optimized according to the treatment plan used.

[0061] In some cases, recombinant viruses (e.g., VSV) provided herein can be administered directly. For example, the virus can be injected directly into a tumor that is palpable through the skin (e.g., a breast cancer tumor). Ultrasound guidance can also be used in this manner. Alternatively, direct administration of the virus can be achieved via a catheter line or other medical access device and can be used in conjunction with an imaging system for locating cancer cell populations. In this method, an implantable administration device is positioned, typically near the cancer cell population, using a guidewire inserted into the medical access device. An effective dose of recombinant virus (e.g., VSV) provided herein can be administered directly to cancer cell populations that can be seen in the exposed surgical field.

[0062] In some cases, recombinant viruses (e.g., VSVs) provided herein can be delivered systemically. For example, systemic delivery can be achieved intravenously by injection or via intravenous delivery devices designed for the administration of multiple doses of the drug. Such devices include, but are not limited to, butterfly infusion needles, peripheral venous catheters, midline catheters, peripherally inserted central catheters, and surgically placed catheters or ports.

[0063] The course of treatment with recombinant viruses (e.g., VSV) provided herein can be monitored by evaluating changes in clinical symptoms or by directly monitoring the number of cancer cells or the size of the tumor. In the case of solid tumors, the effectiveness of viral therapy can be evaluated by measuring the size or weight of the tumor before and after treatment. Tumor size can be determined directly (e.g., using calipers), or by using imaging techniques (e.g., X-ray, magnetic resonance imaging, or computed tomography), or by evaluating non-imaging optical data (e.g., spectral data). For cancer cell populations (e.g., leukemia cells), the effectiveness of viral therapy can be determined by measuring the absolute number of leukemia cells in the patient's blood circulation before and after treatment. The effectiveness of viral therapy can also be evaluated by monitoring the levels of cancer-specific antigens. Cancer-specific antigens include, for example, carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), CA125, alpha-fetoprotein (AFP), glycosylation antigens 15-3, and glycosylation antigens 19-4.

[0064] The present invention will be further illustrated by the following embodiments, but these will not limit the scope of the invention as described in the claims. [Examples]

[0065] Example 1: CDV F and H polypeptides and recombinant virus cell line Vero African green monkey kidney cells (Vero; American type culture collection [ATCC], Cat.# CCL-81) and their derived cell lines (Nectin-4 (Noyce et al., Virology, 436(1):210-20 (2013)), SLAMF1 (Tatsuo et al, Nature, 406(6798):893-7 (2000)), canine SLAMF1 (von Messling et al., J. Virol., 77(23):12579-91 (2003), or membrane-immobilized hexahistidine peptides). (Sequence ID 21)The cells expressing one of the single-chain antibodies specific to (Nakamura et al., Nat. Biotechnol, 23(2):209-14 (2005)) were maintained in Dulbecco's Modified Eagle Medium (DMEM) (GE Healthcare Life Sciences, Cat.# SH30022.01), which was supplemented with 5% (volt / volt) thermally demobilized fetal bovine serum (FBS, Gibco), as well as 0.5 mg / mL Geneticin (G418; Corning) for Vero / NECTIN-4 and Vero / SLAMF1, and 1 mg / mL Zeocin (ThermoFisher) for Vero / dogSLAMF1. Human renal epithelial cells (HEK293T cells) (obtained from Dr. Cosset (Universite de Lyon)), baby hamster kidney cells (BHK, ATCC, Cat.# CCL-10), human glioblastoma U-87 MG cells (ATCC, catalog number HTB-14), and SKOV3ip.1 human ovarian tumor cells were maintained in DMEM + 10% FBS. Chinese hamster ovarian cells (CHO cells), CHO-CD46, and CHO-EGFR (Nakamura et al, Nat. Biotechnol., 22(3):331-6 (2004)), CHO-SLAMF1 (Tatsuo et al., Nature, 406(6798):893-7 (2000)), CHO-dogSLAMF1 (Seki et al., J. Virol, 77(18):9943-50) CHO-NECTIN4 (Liu et al., J. Virol., 88(4):2195-204 (2014)), CHO-CD38 (Peng et al., Blood, 101:2557-62 (2003)), CHO-HER2 / neu (Hasegawa et al., J. Virol., 81(23):13149-57 (2007)), Burkitt B-cell lymphoma Ramos (ATCC, Cat.# CRL-1596), and Raji cells (ATCC, Cat.# CCL-86) were cultured in Roswell Park Memorial Laboratory (RPMI) 1640 medium (Corning Inc, Cat. #10-040-CV, Corning, NY, United States), as otherwise described.

[0066] Construction of plasmids and whole-genome rMeV To construct the CDV 22458 / 16 expression plasmid, total RNA was extracted from Vero / canine SLAMF1 cells (passage 1) infected with the CDV 22458 / 16 isolate using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Both the CDV-H and CDV-F genes were reverse transcribed using SuperScript III reverse transcriptase (Thermo Fisher Scientific, Cat.# 11752050) and amplified by PCR using the following primers: CDVH7050(+):AGAAAACTTAGGGCTCAGGTAGTCC (Sequence ID 22) CDVH8949(-):TCGTCTGTAAGGGATTTCTCACC (Sequence No. 23) CDVF4857(+):AGGACATAGCAAGCCAACAGG (Sequence ID 24) CDVH7050(-):GGACTACCTGAGCCCTAAGTTTTCT (Sequence ID 25)

[0067] The PCR product was directly sequenced by Sanger (Genewiz, Plainfield NJ, USA) and cloned into the pJET1.2 vector (Thermo Fisher). Next, the CDV H open reading frame (Figure 8) was used as a forward primer. [ka] (Sequence ID 26) and reverse primer [ka] (Sequence ID 27) The pCG vector (Cathomen et al., J. Virol., 72(2):1224-34 (1998)) was PCR amplified using [a specific method] and restricted and cleaved with PacI and SpeI (New England Biolabs, Iswich MA, USA), and then cloned using the InFusion HD kit (Takara, Shinagawa, Tokyo, Japan). The primers provided the PacI and SpeI restriction sites (underlined, respectively) and the untranslated region of MeV-H (italicized). Similarly, the CDV F open reading frame (Figure 9; amino acid residues 136-662 of SEQ ID NO: 4) was cloned into the HpaI / SpeI restricted pCG-CDV-F plasmid (von Messling et al., J. Virol., 75(14):6418-27 (2001)). The resulting plasmid pCG-CDV F 22458 / 16 contains the MeV-F untranslated region and the MeV-F signal peptide.

[0068] The CDV H / F Onderstepoort vaccine and isolate 5804 (von Messling et al., J. Virol., 75(14):6418-27 (2001)), as well as the expression plasmid for MeV Nse, are described in other literature (Cathomen et al., J. Virol., 72(2):1224-34 (1998)). The retargeting form of the H protein was constructed by inserting homologous PacI / SfiI digestion PCR products into a pTNH6 vector (Nakamura et al., Nat. Biotechnol., 23(2):209-14 (2005); and Nakamura et al., Nat. Biotechnol., 22(3):331-6 (2004)). Site-directed mutagenesis (QuickChange Site-Directed Mutagenesis Kit, Agilent Technologies, Santa Clara, CA, USA) was used to remove the tropism of H and the SpeI site of CDV-F, thereby introducing truncation to the cytoplasmic terminal.

[0069] Envelope-exchanged rMeV was generated by closing the PacI / SpeI and NarI / PacI regions of the corresponding expression plasmid. rMeV rescue was performed using the START system (Nakamura et al., Nat. Biotechnol., 23(2):209-14 (2005)).

[0070] Protein expression Cells were transfected using Fugene HD (PROMEGA, Fitchburg WI, USA) or TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison WI, USA). For quantitative fusion assays, a dual-split reporter system (Kondo et al., J. Biol. Chem., 285(19):14681-8 (2010); and Ishikawa et al., Protein Eng. Des. Sel., 25(12):813-20 (2012)) was used as otherwise described (Munoz-Alia et al., Viruses, 11(8), pii: E688, doi: 0.3390 / v11080688 (2019)), and BHK cells were used as effector cells. For semi-quantitative evaluation of fusion, Vero cells and derivatives were transfected with 1 μg each of H and F expression plasmids and stained with Hema-Quik (Thermo Fisher Scientific, Cat.#123-745) after 1 day. Images were obtained at 4x magnification using a microscope (Eclipse Ti-S; Nikon). Alternatively, a GFP expression plasmid was incorporated to further visualize syncytium formation. To assess the levels of H polypeptide, transfected cells were analyzed by flow cytometry or CELISA using 6×His-tagged monoclonal antibodies (Miltenyi Biotec, Cat. # 130-120-787 or Thermo Fisher Scientific, Cat. #MA1-135) as otherwise described (Munoz-Alia et al., Viruses, 11(8), pii: E688, doi: 10.3390 / v11080688 (2019); and Saw et al., Methods, 90:68-75 (2015)).To analyze total protein expression by flow cytometry, cells were treated with eBioscience Intracellular Fixation & Permeabilization Buffer (Thermofisher, Cat.#88-8823-88).

[0071] Viral protein content The viral preparation was heated in the presence of dithiothreitol, fractionated onto a 4-12% Bis-Tris polyacrylamide gel, and transferred to a polyvinylidene fluoride membrane. The blot was analyzed using anti-MeV-Hcyt (Cathomen et al., J. Virol., 72(2):1224-34 (1998)), anti-MeV-N (Toth et al., J. Virol., 83(2):961-8 (2009)), or anti-His tag (Genscript, Piscataway NJ, USA, Cat.# A01857-40) antibodies, with a secondary rabbit antibody conjugate (ThermoFisher, Cat.#31642) used as a probe for detection. This blot was incubated with SuperSignal Wester Pico chemiluminescent substrate (ThermoFisher) and analyzed using ChemiDoc Imaging System (Bio-Rad).

[0072] Neutralization assay The fluorescence focus reduction neutralization assay was performed as described elsewhere (Munoz-Alia et al., J. Virol., 91(11): e00209-17 (2017)). Polyclonal anti-canine distemper virus, Lederle Avirulent (antiserum, ferret), was obtained from BEI Resources (NR-4025). Human serum was pooled from 60-80 donors, particularly those with blood type AB (Valley Biomedical Products & Services, Inc, Cat.#HS1017, Lot #C80553).

[0073] result Fabrication of fusion-induced CDV H / F complex The envelope glycoprotein of CDV has 36% (H polypeptide) and 66% (F polypeptide) amino acid homology with that of MeV. Open reading frames for the H and F polypeptides were obtained from the first passage wild-type CDV isolate SPA.Madrid / 22458 / 16 (CDV 22458 / 16) isolated from postmortem tissue of dying dogs. Phylogenetic analysis of the full-length hemagglutinin gene using the maximum likelihood method showed that the CDV H polypeptide of 22458 / 16 is grouped within the Artic clade (Figure 1). Simultaneous transfection of the H / F complex of 22458 / 16 showed fusion activity only in the presence of the SLAMF1 receptor (Figure 2A). Lack of fusion activity was observed in Nectin-4 expressing cells. Co-expression of heterotype wild-type 5804 CDV H polypeptide and 22458 / 16 CDV F polypeptide resulted in significant syncytium formation in Nectin-4 expressing Vero cells. On the other hand, the H / F complex derived from the large plaque-forming variant of the Onderstepoort vaccine strain resulted in significant syncytium formation regardless of Nectin-4 and SLAMF1 expression.

[0074] To more accurately confirm syncytium formation using a reporter gene, heterotype wild-type CDV H 5804 Polypeptides and CDV F 22458 / 16 The above fusion phenotype was confirmed by the combination of polypeptides (Figure 2B). Homotype H / F 5804 The absence of syncytium formation is thought to be due to the presence of a naturally occurring 135-amino acid signal peptide, but CDV H / F induces intercellular fusion only in a SLAMF1-dependent manner. 22458 / 16 Further investigation was conducted into the clearly defined fusion phenotypes observed in CDV H. 22458 / 16 The clone used for the polypeptide was found to contain an amino acid change in the consensus sequence (M437L). Using this clone, CDV H was created by changing the 437th position from leucine to methionine. 22458 / 16We created a clone that codes for polypeptides.

[0075] The expansion of tropicalism in CDV fusion devices Co-transfection of wild-type CDV H / F complexes resulted in specific receptor-dependent syncytium formation, but this was not the case with H / F derived from the Ondertepoort vaccine. Therefore, to determine whether receptor utilization could be extended to other receptors, the following experiment was performed. CD38 was selected as the target receptor, and for this purpose, CD38-specific scFv was displayed on the carboxy-terminal domain of the adherent protein (Figure 2C). For comparison, MeV H polypeptide and Nipah G polypeptide were also included in the analysis. The results shown in Figure 2D demonstrate that different constructs were expressed on the surface at comparable levels. Notably, CDV H 22458 / 16The L437M substitution did not appear to affect cell surface expression. Next, fusion ability was quantitatively compared using a self-associating split phosphate ferase assay described in other literature (Kondo et al., J. Biol. Chem., 285:14681-14688 (2010); and Ishikawa et al., Protein Eng. Des. Sel., 25:813-820 (2012)). In this assay (Figure 2E), effector cells were transfected with expression plasmids encoding the H / F complex and one half of the dual-split GFP / sea urchin phosphate ferase protein (DSP1-7). Similarly, target cells expressing the relevant receptor were transfected with the other half (DSP8-12). Mixing of the contents causes the otherwise non-functional dual-split GFP / sea urchin phosphate ferase proteins to associate, and their activity is measured. The results of this experiment using effector cells expressing different H or G / F complexes are shown in Figure 2F. No fusion activity was observed when using the parental CHO cell line, but fusion activity was evident in CHO cells engineered to stably express HIS-specific scFv (CHO-HIS) or the CD38 molecule. Generally, the activity level was even more pronounced when using CD38 targeting compared to the pseudoreceptor system 6×His-anti-6×HIS scFv. These differences are related to Nipar G αCD38 This was more pronounced when using [specific method / tool]. The former's fusion ability was heterotype CDV H 5804 αCD38 Polypeptide / CDV F 22458 / 16 The levels were significantly lower compared to polypeptides or homotype H / F derived from the Onderstepoort strain. Surprisingly, CDV H 22458 / 16 L437M substitution of the polypeptide dramatically altered the polypeptide fusion phenotype, even when no difference was observed at the expression level (Figure 2G). This novel fusion ability is linked to CDV F 5804Only when the polypeptide signal peptide was replaced with a shorter MeV F signal peptide did it become equivalent to that obtained from homotype CDV H / F from 5804. Nevertheless, CDV H 5804 αCD38 Polypeptide / CDV F 22458 / 16 The fusion level of the polypeptide heterotype combination was comparable to and superior to that obtained with the Onderstepoort large plaque-forming variant. These results indicate that not only can the CDV fusion mechanism be designed to use a different receptor, but that a receptor-dependently fusion-inducible phenotype can also be obtained from heterotype combinations of H / F complexes derived from different CDV strains. This improvement in the fusion ability of the CDV H / F complex was achieved by shortening the signal peptide of the CDV F polypeptide and using a CDV polypeptide containing M437.

[0076] Receptor targeting The CDV H polypeptide described in the paragraph above was still capable of using human Nectin-4 as a receptor. To prevent Nectin-4 interaction, nucleic acids encoding the CDV H polypeptide containing the Y539A mutation were constructed. As shown in Figure 2H, CDV H polypeptide containing the Y539A point mutation αCD38 Polypeptide (CDV H Y539A αCD38 ) lost its fusion activity on human Nectin-4 cells, but CHO-HIS (CDV H Y539A αCD388 and CDV H Y539A ) cells and CHO-CD38 (CDV H Y539A αCD388 It was found that it still possessed the ability to induce fusion on cells. This fusion activity was found in the fully retargeted MeV H polypeptide (MeV Haals). αCD38 and MeV Haals αCD38 The activity was equivalent to that obtained by [method / method]. These results indicate that the CDV H / F complex can be efficiently retargeted to specific receptors.

[0077] Effect of ligand binding affinity on cell-cell fusion driven by CDV H / F To evaluate whether differences in ligand binding affinity displayed on the ectodomain of the CDV H polypeptide affect fusion, Her2 / neu-specific scFv binders and aphibody molecules (Hasegawa et al., J. Virol., 81(23): 13149-57 (2007); Wikman et al., Protein Eng. Des. Sel., 17(5):455-62 (2004); and Orlova et al., Cancer Res., 66(8):4339-48 (2006)) were displayed. Figure 2I shows that binding affinity higher than 1 nM was required to induce fusion on CHO cells engineered to stably express the Her2 / neu molecule, but this was not the case in the parental cell line. This was true regardless of whether the binder was in the form of scFv or aphibody molecules. To investigate the relationship between receptor density and binder affinity, quantitative fusion assays were repeated using arrays of cancer cell lines expressing different levels of Her2 / neu molecules on their surface: HT1080 (1.2 × 10⁻¹⁰). 4 ), Sko3pi (1.5 × 10 5 ), and TET67L (4.3 × 10 3 By displaying the affibody with the highest binding affinity (Z342, 0.022 nM), CDV H was able to induce fusion in all cell lines tested, regardless of receptor density. However, Z4 (50 nM) enabled cell-to-cell fusion only in Skov3pi cells, which expressed the highest receptor density. These results indicate a correlation between binder affinity and receptor density on target cells, suggesting that lower receptor densities are more effective with higher binder affinity.

[0078] CDV H / F complexes targeting CD46 can overcome the neutralization susceptibility of oncolytic measles virus. To obtain scFv-CDV H polypeptides that retain fusion levels similar to those of the MeV H Nse strain, different CD46-specific scFv binders were displayed on CDV H polypeptides. Cell surface expression levels were compared (Figure 3A). Cell enzyme-linked immunosorbent assay (CELISA) showed that both untargeted MeV H polypeptides and CDV H polypeptides, as well as CD46-targeted CDV H polypeptides, were similarly expressed on the cell surface. Next, quantitative fusion assays revealed that only MeV H polypeptides induced fusion activity in CHO-Nectin-4 cells, while all but the untargeted CDV H polypeptides induced fusion in CHO-CD46 cells (Figure 3B). scFv binders A10, A09, G09, and K2 displayed on CDV H polypeptides induced fusion levels similar to those observed with MeV H polypeptides, but scFv G101469 and K01 induced relatively lower levels. To replace the existing MeV envelope, the CDV H-scFv A09 / CDV F polypeptide was selected. This virus, referred to as Stealth 2.0, was rescued, and it was confirmed that the H / F polypeptide was successfully displayed in virions. Western blot analysis confirmed that when an anti-MeV H polypeptide antibody was used against the cytoplasmic end, the MeV H polypeptide was detected only in the case of MeV. In contrast, the CDV H-scFV A09 polypeptide was detected only when the membrane was searched using an anti-6×HIS-tag antibody as a probe (Figure 3C). This same HIS-tag system enabled replication of the Stealth 2.0 virus in Vero cells stably expressing anti-HIS scFv, but not in the parental Vero cell line (Figure 3D). This replication dynamic was equivalent to that obtained with MeV on Vero / hSLAM. In summary, these results indicate that the measles virus envelope H and F polypeptides can be replaced with CDV-derived H and F polypeptides without negatively affecting viral replication.

[0079] The neutralization susceptibility of Stealth 2.0 was examined using serum pooled from 20-30 American donors. CDV antiserum was used as a control. Figure 3E shows that Stealth 2.0 is insensitive to the neutralizing activity of MeV antiserum. In contrast, the neutralization pattern of MeV was essentially the opposite: it was neutralized by anti-measles antibodies but not by anti-CDV antibodies.

[0080] Since viral entry can occur even without apparent fusion, the following experiment was performed to confirm viral tropism induced by a novel envelope. When CHO cells expressed the receptor CD46, Nectin-4, and canine or human SLAMF1, viral GFP autofluorescence was observed (Figure 3F). Conversely, GFP autofluorescence induced by Stealth 2.0 was observed only in the case of anti-6×HIS scFv expression (CHO-HIS), CD46, and canine SLAMF1. These results indicate that human CD46-targeted measles virus resistant to neutralization of anti-measles virus antibodies can be created by using a CDV H / F envelope that targets CD46.

[0081] Stealth 2.0 induces antitumor effects similar to those of parent MeV. To evaluate the use of Stealth 2.0 as an oncolytic agent, the following was performed: SCID mice with U266.B1 tumors were treated with a single intravenous dose of either MeV or Stealth 2.0 (Figure 4A). The transplanted tumors in the PBS-treated group continued to grow exponentially (Figure 4B), and by day 12, all mice had to be euthanized due to tumor tissue volume (Figure 4C). In contrast, tumor progression was slow in both treatment groups, resulting in a significantly increased median survival time. Similar oncolytic activity was observed for Stealth 2.0 and MeV, and since the latter can utilize the receptors Nectin-4 and SLAMF1 in addition to CD46, these results demonstrate that targeting CD46 is sufficient for tumor regression in a multiple myeloma model. Furthermore, Stealth 2.0 was demonstrated to be usable as an alternative to the current oncolytic MeV vaccine when patients have high levels of neutralizing anti-measles virus antibodies.

[0082] The CDV H / F complex can retarget other mononegative virus species. To investigate the compatibility of the CDV H / F complex that controls tropism in VSVs belonging to the Lyssavirus genus of the Rhabdoviridae family, the following was performed: VSV-hIFNβ-NIS (Naik et al., Mol. Cancer Ther., 17(1):316-326 (2018)), a VSV engineered to express interferon-β (IFN-β) and sodium-iodine cotransporter (NIS), was obtained and modified by replacing the VSV-G polypeptide with CDV H and F polypeptides using the technique described in other literature (Ayala-Breton et al., Hum. Gene Ther., 23(5):484-91 (2012)). 22458 / 16 Polypeptides, and parent CDV H 5804Either the polypeptide (VSV-CDVFH-GFP) or a CDV H polypeptide retargeted to the EGFR (VSV-CDVFHaal-αEGFR-GFP) or CD38 (VSV-CDVFHaal-αCD38-GFP) receptor was used (Figure 5). Furthermore, all CDV H polypeptides were modified to eliminate interaction with canine SLAMF1. Y539A The background sample contained the R529A mutation (CDV Haa).

[0083] To confirm that a novel envelope complex governs viral tropism, a panel of CHO cells expressing specific receptors was infected. As shown in Figure 6, when the virus displayed the parental CDV F / H complex, GFP autofluorescence was observed in cells expressing either Nectin-4 or the canine SLAMF1 receptor. In contrast, when EGFR-specific scFv CDV H was present, infection and GFP autofluorescence were observed only in cells expressing the EGFR receptor. Similarly, when CD38-specific scFv CDV H was present, infection and GFP autofluorescence were observed only in cells expressing the CD38 receptor. In this case, syncytium formation and cell death effects were observed in CD38-expressing cells but not in CHO cells expressing the EGFR receptor; on the other hand, the opposite pattern was observed with the EGFR-specific virus (VSV-CDVFHaal-αEGFR-GFP) (Figure 7). These results reveal that the CDV F / H complex can be used to specifically control cell entry and syncytium formation in a receptor-specific manner through scFv display using selected receptors to alter the direction of viral tropism, in association with rhabdoviruses.

[0084] The use of this system as a tumor-lytic vector was also evaluated in vivo (Figure 8). Athymic nude mice with SKOV3ip.1 tumors were treated with a single dose of either EGFR-targeted VSV or VSV-hIFNβ-NIS, which is currently in clinical trials. VSV-hIFNβ-NIS did not improve survival compared to the PBS-treated control group, but EGFR-targeted VSV resulted in a significant improvement in survival (p<0.005). These results demonstrate that the targeted VSV described herein can be used for oncological purposes.

[0085] MeV targeting CD38 and EGFR To confirm that CD38 and EGFR targeting of MeV could be achieved using CDV F and H polypeptides, the following was performed (Figure 11). To enhance safety, additional mutations were inserted into the CDV H polypeptide to prevent a reversion to the use of the innate receptor (Sawatsky et al., J. Virol., 92(15):e0069-18 (2018)). In addition to the R529A amino acid substitution, substitutions of D526A, I527A, S528A, R529A, Y539A, Y547A, and T548 were also incorporated (Figure 11A). Figure 11B shows that the introduction of these mutations did not affect the fusion induction ability of the CD38-targeted CDV H polypeptide. Similarly, Figure 11C shows that these various point mutations did not affect the neutralization sensitivity of the polypeptide to measles-immunized human serum. The fusion activity of the CD38-targeted MeV H polypeptide was inhibited by pooled measles antiserum up to a 1:80 dilution, while the homologous CD38-targeted CDV H polypeptide was not inhibited, even at the highest concentration tested, a 1:10 dilution. Similar to rhabdovirus, measles viruses incorporating the targeted CDV H / F complex were able to infect and fuse with cells specifically targeting CD38 or EGFR (Figure 11D). This infection specificity was also observed in many tumor cell lines. Skov3pi and U87 cells (EGFR-positive) were infected with EGFR-targeted viruses but not with CD38-targeted viruses. In contrast, Raji and Ramos cell lines (CD38-positive) were infected only with CD38-targeted viruses. As a control, 6×HIS pseudoreceptors were used. (Shown as sequence number 21, "6×HIS") Cells expressing the compound were infected with all retargeted viruses. These results demonstrate that the CDV F / H complex can be used to evade anti-measles immunity and lead to targeted entry and syncytium formation in cells.

[0086] The in vivo oncolytic activity of CD38 and EGFR-targeted MeV was also investigated. SKOV3ip.1 tumors were transplanted subcutaneously or intraperitoneally into athymic nude mice, followed by viral treatment via the same route (Figure 11E). CD38-targeted virus showed some therapeutic efficacy, but the efficacy of EGFR-targeted virus was superior (Figure 11F). In contrast, CD38-targeted virus showed no antitumor effect after intraperitoneal administration, while EGFR-targeted virus resulted in complete tumor regression, as observed by a 100% survival rate. Notably, no difference was observed between MeV retargeted with either the MeV H / F complex or CDV H / F. These results indicate that the CDV H / F complex can enhance the tumoricidal properties of oncolytic measles virus without the problem of neutralization by measles-induced neutralizing antibodies.

[0087] Example 2: Further analysis of CD46-specific oncolytic measles virus resistant to neutralization with measles-immunized human serum. In this embodiment, some of the information and results from Example 1 are repeated, and additional results are provided.

[0088] The heterogeneous combination of wild-type CDV glycoproteins promotes cell fusion. The MeV coat was replaced with a different viral coat that allows the virus to evade neutralization by anti-measles antibodies. Wild-type CDV was selected for this purpose. Although there is a CDV strain approved for vaccine use (Onderstepoort strain), this strain can utilize SLAMF1 and Nectin-4, as well as a currently unidentified receptor (Figure 14), making it difficult to modify the viral tropism. Therefore, we focused on the wild-type strain, which is known to interact only with SLAM and Nectin-4.

[0089] To identify the most fusion-promoting CDV H / F glycoprotein pair, the following was performed: Different H / F combinations obtained from 5804P and SPA.Madrid / 16 (hereinafter referred to as 5804 and SPA, respectively) isolates were transiently expressed in Vero cells expressing SLAMF1 or NECTIN4, and the degree of viral protein-induced cell fusion (synthia formation) was qualitatively evaluated. The fusion activity of CDV-H / F pairs was not observed when the 135 aa-signal peptide was maintained in CDV-F (Figure 15A). When replaced with homologous proteins from MeV F, co-expression of H / F proteins from 5804P resulted in cell fusion in SLAMF1 and NECTIN4-expressing cells, while co-expression of H / F proteins from SPA promoted cell fusion only in SLAMF1-expressing Vero cells. On the other hand, co-expression of CDV-H 5804 and CDV-F SPA, rather than the heterogeneous combination of CDV-H SPA and CDV-F 5804, resulted in syncytium formation in SLAMF1 and NECTIN4-expressing Vero cells. The data summarized above provided evidence supporting the fusion deficiency on CDV-H SPA. To begin investigating whether the fusion deficiency of CDV-H SPA in NECTIN4-expressing cells is due to low affinity for the receptor, the fusion phenotype was quantitatively compared using a non-native receptor to standardize conditions for receptor binding affinity. This method involved fusing a His-tagged CD38-specific scFv to the C-terminal domain of the receptor-binding protein and measuring the fusion level in CHO cells encoding either CD38 or a HIS-tagged pseudoreceptor (CHO-αHIS). Since L437 corresponds to a clone-specific mutation not present in other CDV gene groups, the L437M substitution was incorporated into CDV-H SPA (Figure 16).For comparison, retargeted receptor-binding proteins from other viruses were also added: MeV-H and Nipah-G (Bender et al., PLoS Pathog., 12(6):e1005641 (2016); and Nakamura et al., Nat. Biotechnol., 22(3):331-6 (2004)). To add rigor to the comparison, receptor-binding protein expression was first analyzed by Western blotting and flow cytometry, showing no significant effect on protein folding or surface expression (Figures 17A and 17B). When CDV-H / F pairs from SPA proteins were expressed in CHO-C38, only CDV-H SPA with M437L promoted fusion (Figure 15B). Notably, no significant difference in fusion ability was observed between two homotyped CDV-H / F pairs from SPA or 5804P isolates. Surprisingly, the fusion activity induced by the heterogeneous H / F combination CDV-H 5804 / F SPA surpassed the fusion activity achieved by the homogeneous combinations CDV-H / F 5804 and CDV-H / F SPA. While significant fusion levels were observed in CHO-CD38 cells for the retargeted Nipah G / F pair (p<0.0001), these were not significant compared to the fusion levels obtained by the unretargeted CDV H / F OL pair.

[0090] Based on this series of experiments, we selected the highly fusionable CDV-H 5804 / F SPA pair for further investigation and improvement.

[0091] The strength of the CDV H / F interaction is inversely correlated with the efficiency of cell-cell fusion. The enhanced cell fusion observed with the CDV-H 5804 / F SPA pair may be related to low binding avidity at the H / F interface. This was based on the observation that H / F dissociation is essential for the fusion process (Plemper et al., J. Virol., 76(10):5051-61 (2002); and Bradel-Tretheway et al., J. Virol., 93(13) (2019)). To test this hypothesis, the relative strength of differently related CDV-H and F protein combinations was evaluated by co-IP immunoprecipitation assay. To facilitate detection, CDV-F SPAs were fused with FLAG tags that do not affect the bioactivity of the proteins (Figure 18). Results shown in Figure 15C revealed that the presence of the M437L mutation in CDV-H SPA resulted in a weakened affinity of CDV-H SPA to CDV-F SPA. In addition, the affinity of CDV-F SPA for CDV-H 5804 was slightly lower than the affinity of CDV-F SPA for CDV-H SPA (Figure 15C). In summary, these data indicate an inverse correlation between the fusion induction level and the strength of the CDV-H / F interaction.

[0092] The fully retargeted CDV envelope glycoprotein exhibits fusion activity equivalent to that of the MeV glycoprotein. The CDV-H protein described above was still able to use NECTIN4 as a receptor (Figure 15A). To maximize retargeting efficiency, the CDV-H protein needed to be detargeted from this undesirable interaction with human cells. To determine whether the removal of this native tropism would affect cell fusion induced by CDV-H / F binding to non-native receptors, we introduced the Y539A mutation into CDV-H, which is equivalent to the Y543A mutation in MeV-H. This mutation inhibits NECTIN4-dependent fusion (Mateo et al, J. Virol., 87(16):9208-16 (2013)) but does not affect cell surface expression (Sawatsky et al., J. Virol., 86(7):3658-66 (2012)). Next, the fusion ability of CDV-H 5804 (Y539A) 5804 / F SPA was compared to that of a fully retargeted MeV-H / F pair (Nakamura et al, Nat. Biotechnol., 23(2):209-14 (2005)). For this purpose, a quantitative kinetic fusion assay based on a dual-split GFP / luciferase reporter protein was used. The data shown in Figure 15D demonstrate that CD38-targeted CDV-H 5804(Y539A) did not exhibit fusion activity in CHO-NECTIN-4 cells, but induced fusion in CHO-αHIS cells (construct CDV H 5804(Y539A) / F SPA and CDV H 5804(Y539A)αCD38 / F SPA) and CHO-CD38 cells (CDV H 5804(Y539A)αCD38 / F SPA). Since the fusion activity of the NECTIN-4 blinded CDV-H 5804 / F SPA pair was comparable to that obtained with the fully retargeted MeV-H protein, it was concluded that CDV-H 5804(Y539A) can efficiently retarget the CDV-H / F complex to a specific receptor, and this protein was selected for incorporation into the fully retargeted virus.

[0093] Binding affinity determines the efficient retargeting of the CDV H / F complex to CD46. Considering that the selected CDV-H protein could be efficiently retargeted towards CD38 by fusing with a CD38-specific scFv as described above, this protein was then retargeted towards CD46 by displaying a CD46-specific scFv. It was hypothesized that displaying an scFv that recognizes CD46 with sufficiently high binding affinity at the C-terminus of CDV-H would result in CD46-mediated intercellular fusion activity similar to that induced by the MeV H / F complex. To verify this, anti-CD46 scFvs with high affinity for CD46 were identified by evaluating the binding of several different scFv variants isolated from a phage antibody display library to purified CD46 (Figure 19). Surface plasmon resonance technology was applied using a sensor chip with covalently immobilized anti-Fc antibodies. The chimeric Fc-scFv fusion protein was captured on the sensor surface and subsequently examined with soluble CD46 containing SCR1-4 (Figure 20A). Under these assay conditions, the results showed that the affinity constant (Kd) of the chimeric Fc-scFv fusion protein displaying the A09 and K2 fragments was significantly stronger than that of the K01 and N1E fragments (A09>K2>N1E>K2), primarily due to increased association (A09) or decreased dissociation rate (K2) (Table 4, Figure 20B).

[0094] [Table 4]

[0095] To determine whether fusion of scFv to the CDV-H protein can support CD46-dependent fusion, and if so, how CD46 binding affinity affects cell fusion, the following was performed. As a basic approach, quantitative fusion assays were performed on detargeted CDV H [5804 (Y539)] and retargeted CDV-H [5804 (Y539)-scFv] / F SPA pairs and compared with unmodified MeV / F complexes on CHO cells and CHO cells expressing NECTIN4 or CD46. All proteins were expressed at comparable levels (Figure 21). With the exception of scFv K01, all other anti-CD46 scFv allowed the CDV-H / F complex to induce cell-to-cell fusion in CHO-CD46 cells (Figure 20C) and HeLa cell lines with high CD46 expression (Figure 21). Only the MeV-H / F complex induced cell-to-cell fusion in CHO-NECTIN-4 cells.

[0096] From this series of experiments, it was concluded that there is a threshold for binding affinity for intercellular fusion mediated by CD46 via the retargeted CDV H / F complex, and that above this threshold, there is a positive correlation between binding affinity and intercellular fusion.

[0097] CDV envelope glycoproteins that target CD46 are efficiently incorporated into MeV virions depending on their binding affinity and participate in viral entry. To investigate whether high receptor affinity leads to high viral infectivity, the following was performed. To address this issue, a panel of MeVs of the same genotype was constructed, where the MeV coat was replaced with CDV-F SPA, along with CDV-H 5804 (Y539A) displaying CD46-specific low-affinity (K1), medium-affinity (N1E), and high-affinity (A09) scFv (Figure 22A). These “Stealth” viruses were further engineered to express either eGFP or firefly luciferase as a reporter gene and rescued on Vero-αHIS cells. Next, the ability of the Stealth viruses to infect CHO-CD46 cells or producing cell lines (Vero-αHIS) was evaluated. The data shown in Figure 22B demonstrate that all three Stealth viruses efficiently induced syncytium formation on Vero-αHIS cells, but only Stealth-A09 formed syncytium on CHO-CD46 cells. Stealth-N1E produced small clusters of unfused GFP-positive CHO-CD46 cells, but Stealth-K1 clearly did not infect them (Figure 22B). Using luciferase as an infection reporter, CD46-dependent infection by Stealth-K1 was detected, but luciferase levels were significantly lower than those obtained when cells were infected with Stealth-A09 (Figures 22C and 23), indicating that binding to CD46 determines the efficiency of viral entry.

[0098] To further investigate its properties, Stealth-A09 (referred to as Stealth 2.0 in Example 1) was selected based on its excellent CD46-dependent viral entry. Stealth-A09 replicated in Vero-αHIS cells but not in the parental Vero cell line, indicating efficient viral replication via the HIS pseudoreceptor and a lack of interaction with CD46 derived from African green monkeys (Figure 22D). Western blot analysis was performed on viral specimens to estimate the relative particle-to-infectivity ratio of Stealth-A09 to that of native MeV, and the major structural protein (N) was detected, but no significant difference in expression levels was observed (Figure 22E). Analysis of an equivalent number of viral particles revealed that the data suggested similar particle-to-plaque-forming unit (PFU) ratios for the two viruses, indicating efficient uptake of the foreign envelope into virions. Next, we thoroughly examined the receptor-specific tropism of the virus by infecting a panel of CHO cells that stably express either αHIS, CD46, NECTIN-4, or human or canine SLAMF1. As shown in Figure 22F, MeV infects CHO cells expressing either the receptor CD46 and NECTIN-4, as well as canine or human SLAMF1, while Stealth-A09 infects only CHO-αHIS, CHO-CD46, and CHO-dogSLAM cells, indicating that Stealth-A09 is efficiently retargeted to human CD46.

[0099] Consistent with recent reports showing that CDV-H does not bind to human SLAMF1 (Fukuhara et al., Viruses, 11(8) (2019)), no entry of MeV-Stealth-A09 into CHO-hSLAMF1 was observed (Figure 22F). However, adaptation to human SLAMF1 via different CDV strains had been observed (Bieringer et al., PLoS One, 8(3):e57488 (2013)). Therefore, to evaluate the potential of MeV-Stealth to adapt to human SLAMF1, the virus was successively passaged in Vero-hSLAMF1 cells, and viral tropism was analyzed. Infected cells were blindly passaged eight times at 5-day intervals before tropism was examined. This number of passages has already been shown to be sufficient to induce repeated adaptation of measles virus quasi-isekale (Donohue et al., PLoS Pathog., 15(2):e1007605 (2019)). As shown in Figure 22G, at the end of this selective pressure, MeV-Stealth-A09 was still able to induce syncytium formation only in Vero-dogSLAMF1 cells and not in Vero or Vero-hSLAMF1 cells, although individual GPF-positive cells were not observed. Therefore, this data contradicts the potential for adaptation that would enable the use of pathogenic human SLAMF1 receptors.

[0100] In summary, these results indicate that the MeV H / F glycoprotein is interchangeable with the CD46-retargeted CDV-H / F glycoprotein, and that cell entry depends on receptor affinity.

[0101] The oncolytic activity of MeV-Stealth depends on its CD46 binding affinity. To elucidate the antitumor activity of the Stealth virus and the in vivo role of its CD46 binding affinity, the following was performed. For this purpose, athymic mice with peritoneal disseminated SKOV3ip.1 tumors expressing the firefly luciferase gene (SKOV3ip.Fluc) were subjected to saline or 10 6TCID 50 The mice were treated with a single intraperitoneal administration of Stealth-N1E and Stealth-A09 (n=5). Tumor tissue volume was then monitored using in vivo bioluminescence imaging (Figure 24A). By day 7, comparable reductions in tumor tissue volume were observed in animals treated with either Stealth-N1E or Stealth-A09 compared to the control group (Figure 24B). However, the statistical significance of tumor tissue volume reduction was lost by day 21 for Stealth-N1E, whereas this was not the case for the Stealth-A09 group. Comparison of survival curves showed that only MeV Stealth-A09 increased mouse survival compared to the control group. Thus, Stealth-A09 exhibits oncolytic activity that appears to be dependent on its high CD46 affinity.

[0102] MeV-Stealth achieves tumor lysis and extended survival in mice with myeloma and ovarian tumors. To evaluate the superior efficacy of MeV-Stealth-A09 as an oncolytic agent compared to MeV, the following was performed. First, severe combined immunodeficiency (SCID) mice with subcutaneously implanted human myeloma xenografts (derived from U266.B1 cells) were either left untreated (PBS-treated group) or treated with MeV-Stealth or MeV at suboptimal intravenous doses. Tumors in the PBS-treated group continued to grow exponentially, and by day 12, all mice had to be euthanized due to tumor tissue volume (Figure 25A). Treatment with either MeV or MeV-Stealth-A09 slowed tumor progression, resulting in a significant increase in median survival time of 7 and 5 days, respectively (Figure 25B). Histological analysis of the transplanted tumors was performed to assess whether the oncolytic effect was due to viral replication. The results shown in Figure 25C strongly suggested that both viruses could target tumor tissue. These results strongly suggest that Stealth, which targets CD46, induces tumor lysis at a similar level to MeV, which targets both CD46 and SLAMF1, thereby inducing tumor regression in this multiple myeloma model.

[0103] Next, the therapeutic effect of MeV-Stealth-A09 was evaluated as an extension of survival time in the presence of measles-immunized serum. Before commencing this in vivo study, the neutralization sensitivity of recombinant viruses was first evaluated in vitro. The results shown in Figure 26 indicate that MeV-Stealth-A09 is insensitive to the neutralizing activity of measles-immunized human serum, but is completely neutralized by CDV-immunized ferret serum or mouse serum. For MeV, essentially the opposite pattern was observed (Figure 23). Next, SKOV3ip.Fluc cells were transplanted into the peritoneal cavity of athymic nude mice that had been administered either PBS or measles-immunized human serum before a single intraperitoneal injection of MeV or MeV-Stealth (Figure 27A). Both animals that did not receive viral injection and animals treated with MeV in the presence of immune serum showed high bioluminescence activity, which continued to increase over time (Figure 27B), indicating that MeV cannot exert a tumor-lytic effect in a pre-existing immune state. In contrast, Kaplan-Meier survival curves showed that, in the absence of immune serum, both oncolytic virus (OV) treatments significantly extended mouse survival, with median survival times of 37 days for MeV-treated mice and 53 days for Stealth-treated mice, compared to 18 days for control mice (Figure 27C). However, the extension of survival time after MeV treatment was completely negated when mice received measles immune serum, whereas MeV-Stealth treatment still significantly extended survival time without statistically significant difference compared to treatment without immune serum (median survival time of 28 days).

[0104] These results demonstrate that targeting CD46 drives oncolytic activity in both xenograft myeloma models and orthotropic models of ovarian cancer, and that replacement of the MeV envelope by the corresponding CDV-H / F fusion device protects MeV from MeV-immunized human serum.

[0105] Methods and Results cell line Baby hamster kidney cells (BHK, Cat.# CCL-10, ATCC, Manassas, VA, USA), human renal epithelial cells (HEK293T) obtained from Dr. Francois-Loic Cosset (Universite de Lyon), and the human ovarian cancer cell line SKOV3ip.1-Fluc (Mader et al., Clin. Cancer Res., 15(23):7246-55 (2009)) were maintained in Dulbecco's modified Eagle medium (DMEM, Cat.# SH30022.01, GE Healthcare Life, Pittsburg, PA, USA) supplemented with 5% fetal bovine serum (FBS; Cat. #10437-028; Thermo Fisher Scientific, Waltham, MA, USA). Vero African green monkey kidney cells (Vero, ATCC, Cat.#CCL-81) and their derivatives (human NECTIN-4 (Noyce et al., Virology, 436(1):210-20 (2013)), human SLAMF1 (Ono et al., J. Virol., 75(9):4399-401 (2001)), or hexahistidine peptide (6×HIS tag) (Sequence ID 21) Cells expressing a membrane-anchored single-strand variable fragment (scFv) specific to ) (Nakamura et al., Nat. Biotechnol., 23(2):209-14 (2005)) were cultured in DMEM (Cat. # SH30022.01, GE Healthcare Life Sciences) as described elsewhere (Munoz-Alia et al., Viruses, 11(8) (2019)). Vero cells constitutively expressing the canine SLAMF1 molecule (Vero-dogSLAMF1) were prepared by introducing a second-generation lentiviral vector (courtesy of Dr. Lukkana Suksanpaisan [Imanis Life Science, MN, USA]) and selecting puromycin. This lentiviral vector was controlled under the control of a spleen focus-forming virus promoter and contained an N-terminal FLAG tag sequence (DYKDDDD). (Sequence No. 28)This gene encodes a canine codon-optimized SLAMF1 molecule (GenBank NP_001003084.1) that possesses ). Cells were maintained in DMEM supplemented with 5% FBS. Chinese hamster ovary (CHO) cell lines, CHO-CD46 cells, CHO-hSLAMF1 cells, CHO-dogSLAMF1 cells, CHO-NECTIN4 cells, CHO-αHIS cells, CHO-CD38 cells, and human myeloma cell line U266.B1 (courtesy of Dr. David Dingli [Mayo Clinic, Rochester, MN]) were grown in RPMI1640 medium supplemented with 10% FBS. Cells were cultured at 37°C in 5% CO2 with saturated humidity.

[0106] Construction of plasmids and whole-genome recombinant measles virus (MeV) To construct a canine distemper virus (CDV) SPA.Madrid / 22458 / 16 expression plasmid, total RNA was extracted from Vero / canine SLAMF1 cells (passage 1) infected with a CDV SPA.Madrid / 22458 / 16 isolate using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Both the CDV-hemagglutinin (H) and CDV-fusion (F) genes were reverse transcribed using SuperScript III reverse transcriptase (Cat.# 11752050, Thermo Fisher Scientific) and amplified by PCR using the following primer: CDVH7050(+): 5'-AGAAACTTAGGCTCAGTAGTCC-3' (Sequence ID 22) ;CDVH8949(-): 5'-TCGTCTGTAAGGGATTTCTCACC-3' (Sequence No. 23) ;CDVF4857(+): 5'-AGGACATAGCAAGCCAACAGG-3' (Sequence ID 24) and CDVH7050(-): 5'-GGACTACCTGAGCCTAAGTTT-3' (Sequence ID 25)The PCR product was directly sequenced using the Sanger method (Genewiz, Plainfield NJ, USA) and cloned into the pJET1.2 vector (Thermo Fisher Scientific). Next, the open reading frame of CDV-H was used as a forward primer. [ka] (Sequence ID 26) and reverse primer [ka] (Sequence ID 27)The pCG vector (Cathomen et al., J. Virol., 72(2):1224-34 (1998)) was PCR amplified using [a specific method] and restricted and cut with PacI and SpeI (New England Biolabs, MA, USA), and then cloned using the InFusion HD kit (Takara, Shinagawa, Tokyo, Japan). The primers contained the PacI and SpeI restriction sites (underlined), as well as the coding sequence of the untranslated region of MeV-H (italicized). Similarly, the open reading frame of CDV-F (amino acid residues 136-662) was cloned into the HpaI / SpeI restricted pCG-CDV-F plasmid (von Messling et al., J. Virol., 75(14):6418-27 (2001)). The obtained plasmid pCG-CDV-F SPA.Madrid / 22458 / 16 contained the coding sequences for the MeV-F untranslated region and signal peptide. Expression plasmids for the CDV-H / F Onderstepoort vaccine and 5804P isolate (von Messling et al., J. Virol., 75(14):6418-27 (2001)) and MeV Nse strain have been described elsewhere (Cathomen et al., J. Virol., 72(2):1224-34 (1998)). The signal peptide of CDV-F 5804 was replaced with heterologous MeV-F, as described above for CDV-F SPA.Madrid / 22458 / 16. Open reading frames for the Nipah-G and Nipah-F glycoprotein genes were amplified from a purchased RNA template (Cat. # NR-37391, BEI Resources), and the Nipah-F gene (GenBank AF212302.2) was inserted into a pCG vector using NarI and PacI sites. Retargeted versions of the H / G protein were created by inserting homologous PacI / SfiI digested PCR products into pCGHX α-CD38 (Peng et al., Blood, 101(7):2557-62 (2003)).Insertion of the CD46-recognizing scFv coding sequence was performed by replacing the anti-CD38 scFv via the SfiI and NotI restriction sites. Site-directed mutagenesis (QuickChange Site-Directed Mutagenesis Kit, Agilent Technologies, Santa Clara CA, USA) was used to remove the H tropism and delete the CDV-F SpeI site.

[0107] The virus used in this example was obtained from a cDNA molecular clone of the Moraten / Schwart vaccine strain pB(+)MVvac2(ATU)P, which has an additional transcription unit downstream of the phosphorylated protein gene (Cathomen et al., J. Virol., 72(2):1224-34 (1998); and Munoz-Alia et al., Viruses, 11(8) (2019)). To avoid plasmid instability during bacterial growth and to enhance viral rescue efficiency, the plasmid backbone was replaced with a pSMART LCkan vector (Cat. # 40821-1; Lucigen, Middleton, WI, USA) having an optimized T7 promoter followed by a self-cleaving hammerhead ribozyme (Hrbz) (Beaty et al, mSphere, 2(2) (2017); and Munoz-Alia et al., Viruses, 11(8) (2019)). eGFP or firefly luciferase was cloned into infectious clones using a unique MluI / AatII restriction site. rMeV rescue was performed using the START system (Nakamura et al, Nat. Biotechnol., 23(2):209-14 (2005)).

[0108] Recombinant protein expression Plasmids encoding CD46-Fc fusion proteins were constructed by fusing the CD46 ectodomain (residues 35-328) with the IgG1 Fc domain (pfc1-hg1e3; InvivoGen, San Diego, CA, USA). scFv K1, K2, and A09 were linked to the VL and VH sequences using the GSSGGSSSG flexible linker. (Sequence ID 29) The sequences were separated, designed, codon-optimized, synthesized, and cloned into pUC57-Kan (GenScript). The fourth scFv(N1E) had its VH and VL sequences linked using the SSGGGS linker. (Sequence ID 30) The DNA was isolated, designed, codon-optimized, synthesized by Creative Biolabs (Shirley, NY), and cloned into pCDNA3.1+ (Invitrogen). For the IgG construct, the scFv was tagged with a 6×HIS tag following the coding sequence of the secretory signal and the C-terminal human Fc region. (Sequence ID 21)The recombinant protein was cloned into the unique AgeI and KpnI sites of pHL-FcHIS (Cat.# 99846, Addgene, Cambridge, MA, USA), which possesses the recombinant protein. The recombinant protein was expressed by transfecting Expi293F suspension cells in serum-free Expi293 expression medium (Thermo Fisher) in a shaker flask according to the manufacturer's instructions. The culture supernatant containing the recombinant protein was collected and passed through a Protein G chromatography cartridge (Cat.# 89926, ThermoFisher). The bound recombinant protein was eluted with 0.1 M glycine (pH 2.0), followed immediately by neutralization with 1 M Tris (pH 8.0), and the separated protein was concentrated using an Amicon Ultra centrifugal concentrator (Millipore Sigma, Burlington, MA, USA). CD46 and NECTIN4 were released from the Fc region by incubation with HRV 3C Protease (Thermo Fisher) at a ratio of 1:200. The final purification process was performed using a Superdex 75 10 / 300 gel filtration column (GE Healthcare) equilibrated with phosphate-buffered saline (PBS). Protein concentration was calculated from the extinction coefficient of the protein, which was determined from the amino acid composition.

[0109] Fusion assay Cells were transfected using Fugene HD (PROMEGA, Fitchburg WI, USA) or TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison WI, USA). For quantitative fusion assays, a dual-split reporter system (Kondo et al., J. Biol. Chem., 285(19):14681-8 (2010); and Ishikawa et al., Protein Eng. Des. Sel., 25(12):813-20 (2012)) was used with BHK cells as effector cells, as otherwise described (Munoz-Alia et al., Viruses, 11(8) (2019)). For semi-quantitative evaluation of fusion, Vero cells and derived cell lines were transfected with a total of 0.1 μg of DNA (H and F expression plasmids in a 1:1 ratio) including a GFP expression plasmid for further visualization of syncytium formation. Images were acquired using a microscope (Eclipse Ti-S; Nikon) at 40x or 100x magnification.

[0110] Morbillivirus adhesion protein expression analysis To assess H polypeptide levels, transfected cells were analyzed by flow cytometry or cell-enzyme-linked immunosorbent assay (CELISA) using anti-6×HIS tag monoclonal antibodies (Cat. # 130-120-787, Miltenyi Biotec, or Cat. # MA1-135, Thermo Fisher Scientific), as described elsewhere (Munoz-Alia et al., Viruses, 11(8) (2019); and Saw et al., Methods, 90:68-75 (2015)). For analysis of total protein expression by flow cytometry (FACSCanbt, BD Biosciences, San Jose, CA, USA), cells were treated with eBioscience Intracellular Fixation & Permeabilization buffer (Cat. # 88-8823-88, Thermo Fisher Scientific).

[0111] Immunoprecipitation (co-IP) of envelope glycoproteins Three micrograms (1 μg of H and 2 μg of F) of total DNA were transfected into HEK293T cells (4e5 cells). After 24 hours, the cells were washed twice with PBS, treated with 1 mM crosslinking agent 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP; Cat.# 21578, Thermo Fisher Scientific), then quenched with 20 mM Tris / HCl (pH 7), and lysed with 0.4 mL M-PER mammalian protein extraction reagent (Thermo Fisher Scientific) containing a 1× Halt protease and phosphatase inhibitor cocktail (Cat. # 1861281, Thermo Fisher Scientific). The soluble fraction was collected after centrifugation at 10,000×g at 4°C for 10 minutes, and 1 / 30th of this amount was set aside as cell lysate input. The remainder was incubated with 0.5 μg of anti-FLAG monoclonal antibody M2 (Sigma-Aldrich) and EZview red protein G affinity gel (Sigma-Aldrich, St. Louis, MO, USA). The precipitate was washed (20 mM Tris-HCl, pH 7.4, 140 mM sodium chloride) and denatured by boiling in laemmli buffer containing β-mercaptoethanol.

[0112] SDS-PAGE and Immunoblotting The samples were fractionated by gel electrophoresis on 4-12% NuPAGE Bis-tris gels (Thermo Fisher) and transferred to polyvinylidene fluoride (PVDF) membranes using the iBLOT 2 dry blotting system (Cat. # IB21001, Thermo Fisher Scientific). The protein materials include antibodies anti-MeV-H606 (Hudacek et al., Cancer Gene Therapy, 20(2):109-16 (2013)), anti-MeV-F431 (von Messling et al., J. Virol., 78(15):7894-903 (2004)), and anti-Fcyt (von Messling et al., J. Virol., 75(14):6418-27 (2001)), anti-MeV-N (Toth et al., J. Virol., 83(2):961-8 (2009)), anti-HIS (Cat.# A01857-40, GenScript, Piscataway NJ, USA), anti-β-actin (Cat.# A3854, Sigma-Aldrich), and anti-CD46 (Cat.# Detection was performed by incubation with sc-7056 (Santa Cruz, Dallas TX, USA). Immunoblots were visualized using rabbit horseradish peroxidase (HRP)-labeled secondary antibody and KwikQuant Imager (Kindle Bioscience LLC, Greenwich CT, USA). Representative results from two independent replicated experiments are shown. Band quantification was performed using KwikQuant Image Analyzer 1.4 (Cat. # D1016, Kindle Biosciences, LLC).

[0113] Antibody binding assay The binding of scFv to CD46 was measured using enzyme-linked immunosorbent assay (ELISA). A Nunc-Immuno MicroWell 96-well solid-phase plate was coated overnight at 4°C with 1 μg of purified CD46 or N4 in 0.05 M carbonic acid-bicarbonate buffer, pH 9.6 (Cat. # E107, Bethyl Laboratories, Montgomery TX, USA). Purified scFv-Fc fusion protein was then diluted in PBS and added at a concentration of 12.5 μg / mL. The bound antibody was detected using a secondary antibody, anti-human IgG (Fc-specific) HRP-labeled antibody (1:70,000; Cat. # A0170, Sigma-Aldrich). In parallel, 125 ng of scFv-Fc fusion protein was first bound to the wells, incubated with the secondary antibody alone, and the protein level was monitored by measuring the optical density (OD 490 nm).

[0114] Surface plasmon resonance (SPR) The interactions between scFv A09, 2B10, K1, K2, and CD46 were measured using a Series S CM5 sensor chip on a Biacore T-100 system (GE Healthcare, Waukesha, WI, USA). For A09, N1E, K1, and K2, 50 μg / mL of anti-FC antibody (MAB1302, EMD Millipore, Burlington, MA, USA), diluted in 10 mM sodium acetate, pH 4.5, was immobilized on the active and reference channels of the CM5 chip using an amine coupling kit reagent (EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide), NHS (N-hydroxysuccinimide), and ethanolamine). Antibody immobilization resulted in approximately 12,000 response units. The interaction between CD46 and anti-FC antibody-captured scFv was measured at 25°C at a data rate of 10 Hz using HBS EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20). Each binding cycle was initiated by loading 15 μg / mL of scFv into the active channel at a flow rate of 10 μL / min for 300 seconds. After a 100-second buffer wash and a 120-second stabilization period, CD46 (concentration range: A09 50 nM-1000 nM, K2 37.5 nM-100 nM, N1E and K1 50-1000 nM) was flowed over the active channel and reference channel at a flow rate of 40 μL / min for 100 seconds. Following the binding phase, a 200-second dissociation period was observed, after which 10 mM glycine pH 2.0 was injected at a flow rate of 30 μL / min for 60 seconds to regenerate the surface-immobilized anti-FC antibody. All sensograms were fitted using a 1:1 binding model with Biacore T100 evaluation software v2.04.

[0115] Analysis of infection and viral replication dynamics For viral infection, cells were infected in Opti-MEM I serum-reduced medium at 37°C for 90 minutes at the specified MOI. After the absorption stage, the inoculum was removed, washed, and viral growth medium (DMEM + 5% FBS) was added. When eGFP-expressing virus was used, fluorescence microscopy images were taken 48 hours after infection. For infection with fluc-expressing virus, 0.5 mM D-luciferin was added to infected cells, and luciferase expression was measured using an Infinite M200 Pro multimode microplate reader (Tecan Trading AG).

[0116] For viral replication dynamics analysis, Vero cells and derived cell lines seeded in 6-well plates 16–18 hours prior to infection were infected in Opti-MEM (Cat. # 31985070, Thermo Fisher Scientific) for 90 minutes at a multiple of infection (MOI) of 0.03. Afterward, the inoculum was removed, and the cell monolayer was washed three times with Dulbecco's phosphate-buffered saline (DPBS; Cat.# MT-21-031-CVRF, Mediatech, Inc, Manassas, VA, USA). The culture medium was then replaced with 1 mL of DMEM supplemented with 5% FBS. At the indicated time, the cell supernatant was collected, cells were scraped out and placed in 1 mL of Opti-MEM, followed by three freeze / thaw cycles. Cell debris was removed by centrifugation (2,000 xg, 5 minutes), and viral titers in Vero-αHIS cells were measured.

[0117] Immunotherapy Male and female HuCD46Ge-IFNARKO mice aged 4-6 weeks (Mrkic et al., J. Virol., 74(3):1364-72 (2000)) lacking type I IFN receptors and expressing human CD46 through gene transfer were subjected to 1 × 10⁻¹⁴ doses. 5 TCID 50 Particles of MeV or Stealth-A09 were inoculated intraperitoneally (ip). Serum samples were collected on day 28 and stored at -20°C until neutralizing antibodies were evaluated.

[0118] Neutralization assay The fluorescence-based plaque reduction microneutralization (PRMN) method was performed as described elsewhere (Munoz-Alia et al., J. Virol., 91(11) (2017)). Briefly, Vero-αHIS cells were seeded in a 96-well plate, and serial dilutions of serum samples were pre-mixed with the virus inoculum at 37°C for 1 hour before being added to the cells. The data were plotted as log (serum dilution) versus standardized reaction (variable slope) using GraphPad software (Prism 8), and the 50% neutralization dose was calculated (ND50). By including the 3rd World Health Organization International serum standard (3 IU / mL), it became possible to convert antibody titers to mIU / mL by calculating a unit-defined constant (Haralambieva et al., Vaccine, 29(27):4485-91 (2011)). Human serum pooled from 60-80 donors with blood type AB (Cat. # HS1017; Lot # C80553, Valley Biomedical Inc, Winchester, VA, USA) was used. The following reagents were obtained from the NIH Defense and Emerging Infections Research Resources Repository, NIAID, NIH: polyclonal anti-MeV antibody, Edmonston (antiserum, guinea pig), NR-4024, and polyclonal anti-CDV Lederle Avirulent (antiserum, ferret), NR-4025.

[0119] We assessed the lack of cross-neutralization between measles virus and Stealth (Figure 28A-B).

[0120] Experimental tumor lysis treatment To establish subcutaneous tumors, 1 × 10⁶ female mice with severe combined immunodeficiency (SCID) at 6 weeks of age were given 1 × 10⁶ 7 1 x 10¹ U266.B1 tumor cells were injected into the right flank. When the tumor reached a diameter of 0.5 cm, 1 x 10¹ mice were given a 2¹⁶ U266.B1 tumor cell injection.5 50% of the infectious dose of tissue culture cells (TCID) 50 A single intravenous dose of MeV (n=5) or Stealth (n=5) was administered to control mice (n=5). Equal amounts of PBS were injected into the control mice. The animals were euthanized when the tumor ulcerated or when the tumor volume reached 20% of body weight. The diameter of the tumor was measured every other day, and the tumor volume was calculated using the formula length × length × width × 0.5.

[0121] To establish an orthotopic model of ovarian cancer, 5×10⁶ cells expressing firefly luciferase (SKOV3ip.1-Fluc) were used. 6 SKOV3ip.1 cells were injected intraperitoneally into athymic nude mice. Ten days later, 600 mIU of measles-immune serum (Cat. # HS1017; Lot # C80553, Valley Biomedical Inc.) or an equal volume of saline was administered, followed by a single intraperitoneal dose of MeV (n=5) or Stealth (n=5) three hours later (1 × 10⁶). 6 TCID 50 The following procedure was performed. The control group of mice was administered the same amount of Vero cell lysate (n=5). In the treatment experiment, instead, 5 × 10 6 Each mouse was transplanted with SKOV3ip.1-Fluc cells. Tumor volume was monitored weekly using in vivo bioluminescence imaging with an IVIS Spectrum instrument (Perking Elmer, Waltham, MA, USA). Mice were euthanized at the end of the study (80 days) when they developed ascites or lost 20% of their body weight. Statistical comparisons between groups were performed using the log-rank test (Mantel-Cox), with p<0.05 considered statistically significant. statistical analysis

[0122] Statistical analysis was performed using GraphPad Prism version 8.3.1 for Mac OS X. Significant differences between groups were determined by one-way analysis of variance (ANOVA) using the Holm-Sidak multiple comparison test. Survival rate data were analyzed using the Kaplan-Meier method, and significant differences between groups were confirmed using the log-rank test.

[0123] Other Embodiments The present invention is described in detail, but it should be understood that the above description is intended to illustrate, and not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages and modifications are within the scope of the claims below. The present invention encompasses the following embodiments. [1] CDV F polypeptide having a signal peptide sequence of less than 75 amino acid residues in length. [2] The CDV F polypeptide according to [1], wherein the signal peptide sequence comprises 75 or fewer amino acid residues of SEQ ID NO: 6. [3] The CDV F polypeptide according to [1] or [2], wherein the CDV F polypeptide is devoid of at least amino acid residues 1 to 60 of SEQ ID NO: 4, or devoid of at least amino acid residues 1 to 105 of SEQ ID NO: 4. [4] The CDV F polypeptide according to any one of [1] to [3], wherein the recombinant virus comprising the CDV F polypeptide and the CDV H polypeptide exhibits increased fusion activity compared to an equivalent control recombinant virus comprising the full-length wild-type CDV F polypeptide and the CDV H polypeptide. A nucleic acid molecule encoding the CDV F polypeptide described in any one of [5][1]~[4]. A recombinant virus containing the CDV F polypeptide described in any one of [6][1] to [4]. Recombinant viruses containing nucleic acid molecules as described in [7][5]. [8] CDV H polypeptide comprising 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, 548A, or combinations thereof, according to the amino acid numbering of Sequence ID No. 2. [9] The CDV H polypeptide according to [8], wherein the CDV H polypeptide comprises a combination of two, three, four, five or six of 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, and 548A.

[10] The CDV H polypeptide according to [8], wherein the CDV H polypeptide comprises a combination of 7, 8, 9, 10 or 11 of 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, and 548A.

[11] The CDV H polypeptide according to [8], wherein the CDV H polypeptide comprises a combination of 12, 13, or 14 of 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, and 548A.

[12] The CDV H polypeptide according to [8], wherein the CDV H polypeptide comprises 454A, 464A, 479A, 494A, 510A, 520A, 525A, 526A, 527S, 528A, 529A, 537A, 539A, 547A, and 548A.

[13] The CDV H polypeptide according to [8], wherein the CDV H polypeptide comprises M437 according to the amino acid numbering of SEQ ID NO: 5.

[14] A CDV H polypeptide having the sequence shown in Figure 11, except that the sequence includes mutations of the presented amino acid residues, selected from the group consisting of P454, V / L / F460, L / F / W479, I494, I / L / V510, Y520, Y / N525, D / G526, I / V527, S / T528, R529, Y / D537, Y539, Y / F547, and T / M548 according to the amino acid numbering of Sequence ID No. 5.

[15] The CDV H polypeptide according to

[14] , wherein the CDV H polypeptide comprises mutations of 2, 3, 4, 5, or 6 presenting amino acid residues selected from the group described above.

[16] The CDV H polypeptide according to

[14] , wherein the CDV H polypeptide comprises mutations of 7, 8, 9, 10, or 11 presenting amino acid residues selected from the group described above.

[17] The CDV H polypeptide according to

[14] , wherein the CDV H polypeptide comprises mutations of 12, 13, or 14 presenting amino acid residues selected from the group described above.

[18] The CDV H polypeptide according to

[14] , wherein the CDV H polypeptide comprises mutations in the group of presenting amino acid residues described above.

[19] The CDV H polypeptide according to

[14] , wherein the CDV H polypeptide comprises M437 according to the amino acid numbering of SEQ ID NO: 5. A nucleic acid molecule encoding the CDV H polypeptide described in any one of

[20] [8]~

[19] . A recombinant virus containing the CDV H polypeptide described in any one of

[21] [8]~

[19] .

[22] The recombinant virus according to

[21] , comprising the CDV F polypeptide described in any one of [1] to [4]. Recombinant viruses containing nucleic acid molecules as described in

[23]

[20] .

[24] The recombinant virus according to

[23] , wherein the virus comprises the nucleic acid molecule described in [5].

[25] The recombinant virus according to any one of [6], [7], and

[21] to

[24] , wherein the recombinant virus is (a) CDV and (b) a hybrid virus of VSV, MeV, or adenovirus.

[26] A virus comprising an RNA molecule, wherein the RNA molecule comprises a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV N polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV P polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV M polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV F polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV H polypeptide, and a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV L polypeptide, but the RNA molecule lacks a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a functional VSV G polypeptide.

[27] The virus described in

[26] , wherein the CDV F polypeptide is the CDV F polypeptide described in any one of [1] to [4].

[28] The virus according to

[26] or

[27] , wherein the CDV H polypeptide is the CDV H polypeptide described in any one of [8] to

[19] .

[29] The virus according to any one of

[26] to

[28] , wherein the CDV H polypeptide comprises the amino acid sequence of a single-chain antibody.

[30] The virus according to

[29] , wherein the single-chain antibody is a single-chain antibody against CD19, CD20, CD38, CD46, EGFR, αFR, HER2 / neu, or PSMA.

[31] The virus according to any one of

[26] to

[30] , wherein the RNA molecule comprises a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a NIS polypeptide. A composition comprising the virus described in any one of

[32] [6], [7], and

[21] to

[31] .

[33] A nucleic acid strand comprising a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV N polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV P polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV M polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV F polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV H polypeptide, and a nucleic acid strand that serves as a template for a positive-strand transcript encoding a VSV L polypeptide, wherein the nucleic acid strand lacks a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a functional VSV G polypeptide, wherein the nucleic acid strand comprises the nucleic acid strand.

[34] The nucleic acid molecule according to

[33] , wherein the CDV F polypeptide is the CDV F polypeptide described in any one of [1] to [4].

[35] The nucleic acid molecule according to

[33] or

[34] , wherein the CDV H polypeptide is the CDV H polypeptide described in any one of [8] to

[19] .

[36] The nucleic acid according to any one of

[33] to

[35] , wherein the CDV H polypeptide comprises the amino acid sequence of a single-chain antibody.

[37] The nucleic acid molecule according to

[36] , wherein the single-chain antibody is a single-chain antibody against CD19, CD20, CD38, CD46, EGFR, αFR, HER2 / neu, or PSMA.

[38] The nucleic acid molecule according to any one of

[33] to

[37] , wherein the RNA molecule comprises a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a NIS polypeptide. A composition comprising a nucleic acid molecule as described in any one of

[39] [5],

[20] , and

[33] to

[38] .

[40] A method for treating cancer, comprising administering a composition according to either

[32] or

[39] to a mammal having cancer cells, wherein the number of cancer cells in the mammal decreases after administration.

[41] The method according to

[40] , wherein the mammal is a human.

[42] The method according to

[40] or

[41] , wherein the cancer is myeloma, melanoma, glioma, lymphoma, mesothelioma, lung cancer, brain tumor, gastric cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, ovarian cancer, breast cancer, pancreatic cancer, liver cancer, or head and neck cancer.

[43] A method for causing tumor regression in a mammal, the method comprising administering a composition according to either

[32] or

[39] to a mammal having a tumor, wherein the size of the tumor is reduced after administration.

[44] The method according to

[43] , wherein the mammal is a human.

[45] The method according to

[43] or

[44] , wherein the cancer is myeloma, melanoma, glioma, lymphoma, mesothelioma, lung cancer, brain tumor, gastric cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, ovarian cancer, breast cancer, pancreatic cancer, liver cancer, or head and neck cancer.

[46] A method for rescuing a reproducible vesicular stomatitis virus from cells, wherein the vesicular stomatitis virus comprises an RNA molecule containing a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV N polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV P polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV M polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV F polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV H polypeptide, and a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV L polypeptide, wherein the RNA molecule lacks a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a functional VSV G polypeptide, and the method is: (a) Inserting nucleic acids encoding RNA molecules into the cells under conditions in which a viable vesicular stomatitis virus is produced, and (b) To recover the vesicular stomatitis virus having the replication ability described above, Methods that include...

Claims

1. A composition for use in treating cancer, wherein the composition comprises a reproducible bullous stomatitis virus comprising an RNA molecule, wherein the RNA molecule comprises a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV N polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV P polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV M polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV F polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV H polypeptide comprising R529A and Y539A according to the amino acid numbering of Sequence ID No. 5 and containing the amino acid sequence of a single-chain antibody, and a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV L polypeptide, but the RNA molecule lacks a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a functional VSV G polypeptide.

2. Recombinant measles virus (MeV), wherein the MeV coat is replaced with CDV H polypeptide and CDV F polypeptide. The CDV H polypeptide contains the amino acid sequence shown in Sequence ID No. 5, except that it contains the respective mutations D / G526, I / V527, S / T528, R529, Y / F547, and T / M548 according to the amino acid numbering of Sequence ID No. 5, and The CDV H polypeptide contains M437 according to the amino acid numbering of SEQ ID NO:

5. The CDV H polypeptide comprises the amino acid sequence of a single-chain antibody, The CDV H polypeptide is characterized by reduced or eliminated tropism against the SLAMF1 polypeptide and / or Nectin-4 polypeptide. virus.

3. The virus according to claim 2, wherein the single-chain antibody is a single-chain antibody against CD19, CD20, CD38, CD46, EGFR, αFR, HER2 / neu, or PSMA.

4. The virus according to any one of claims 2 to 3, wherein the virus comprises an RNA molecule, and the RNA molecule comprises a nucleic acid sequence that serves as a template for a positive-strand transcript encoding an NIS polypeptide.

5. Use of a nucleic acid molecule encoding a CDV H polypeptide for the manufacture of a composition used to treat cancer, wherein the CDV H polypeptide comprises the amino acid sequence shown in SEQ ID NO: 5, except that it contains the respective mutations D / G526, I / V527, S / T528, R529, Y / F547, and T / M548 according to the amino acid numbering of SEQ ID NO: 5, and the CDV H polypeptide comprises M437 according to the amino acid numbering of SEQ ID NO:

5. The CDV H polypeptide comprises the amino acid sequence of a single-chain antibody, The CDV H polypeptide is characterized by reduced or eliminated tropism against the SLAMF1 polypeptide and / or Nectin-4 polypeptide. use.

6. Use of a recombinant virus comprising CDV H polypeptide for the manufacture of a composition used to treat cancer, wherein the CDV H polypeptide comprises the amino acid sequence shown in SEQ ID NO: 5, except that it comprises the respective mutations D / G526, I / V527, S / T528, R529, Y / F547, and T / M548 according to the amino acid numbering of SEQ ID NO: 5, and the CDV H polypeptide comprises M437 according to the amino acid numbering of SEQ ID NO:

5. The CDV H polypeptide comprises the amino acid sequence of a single-chain antibody, The CDV H polypeptide is characterized by reduced or eliminated tropism against the SLAMF1 polypeptide and / or Nectin-4 polypeptide. use.

7. The use according to claim 6, wherein the recombinant virus is (a) CDV and (b) a hybrid virus of VSV, MeV, or adenovirus.

8. A composition comprising the virus according to any one of claims 2 to 4.

9. A composition for use in a method for treating cancer, wherein the method comprises administering the composition to a mammal having cancer cells, the number of cancer cells in the mammal decreases after administration, according to claim 8.

10. The composition according to claim 9, wherein the mammal is a human.

11. The composition according to claim 9 or 10, wherein the cancer is myeloma, melanoma, glioma, lymphoma, mesothelioma, lung cancer, brain tumor, gastric cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, ovarian cancer, breast cancer, pancreatic cancer, liver cancer, or head and neck cancer.

12. A method for in vitro rescuing a reproducible vesicular stomatitis virus from cells, wherein the vesicular stomatitis virus comprises an RNA molecule containing a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV N polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV P polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV M polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV F polypeptide, a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a CDV H polypeptide, and a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a VSV L polypeptide, wherein the RNA molecule lacks a nucleic acid sequence that serves as a template for a positive-strand transcript encoding a functional VSV G polypeptide, and the method is: (a) Inserting nucleic acids encoding RNA molecules into the cells under conditions in which a viable vesicular stomatitis virus is produced, and (b) Recovering the vesicular stomatitis virus having the replication ability described above, Includes, The CDV H polypeptide contains R529A and Y539A according to the amino acid numbering of SEQ ID NO: 5, and also contains the amino acid sequence of a single-chain antibody, or The CDV H polypeptide contains the amino acid sequence shown in Sequence ID No. 5, except that it contains the respective mutations D / G526, I / V527, S / T528, R529, Y / F547, and T / M548 according to the amino acid numbering of Sequence ID No. 5, and The CDV H polypeptide contains M437 according to the amino acid numbering of SEQ ID NO:

5. The CDV H polypeptide comprises the amino acid sequence of a single-chain antibody, The CDV H polypeptide is characterized by reduced or eliminated tropism against the SLAMF1 polypeptide and / or Nectin-4 polypeptide. method.

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